Plant Genome Diversity Volume 2

.
Ilia J. Leitch
Editor-in-chief

Johann Greilhuber • Jaroslav Doležel •

Jonathan F. Wendel
Editors

Plant Genome Diversity

Volume 2
Physical Structure, Behaviour
and Evolution of Plant Genomes
Editor-in-chief
Ilia J. Leitch
Jodrell Laboratory
Royal Botanic Gardens,
Kew Richmond, Surrey
United Kingdom

Editors
Johann Greilhuber Jaroslav Doležel
Department of Systematic and Evolutionary Botany Institute of Experimental Botany ASCR
Faculty of Life Sciences Centre of the Region Hana for
University of Vienna Biotechnological and Agricultural
Vienna Research
Austria Olomouc
Czech Republic

Jonathan F. Wendel
Department of Botany
Iowa State University
Ames, Iowa
USA

ISBN 978-3-7091-1159-8 ISBN 978-3-7091-1160-4 (eBook)

DOI 10.1007/978-3-7091-1160-4
Springer Wien Heidelberg New York Dordrecht London

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Preface

Ever since the origin of life, the evolution of living organisms and their hereditary information
has been accompanied by the development of genetic machinery capable of storing, utilizing,
and transmitting this information between generations. Importantly, this machinery has had to
be flexible, able to respond to the environment and evolve. A characteristic feature of the
genetic machinery in eukaryotes is the partitioning of the hereditary information into smaller
portions—chromosomes. Indeed, the appearance of linear chromosomes was one of the great
evolutionary inventions and paved the way for the formation of large and complex genomes, in
plants as well as in animals. Any consideration of plant genome structure, evolution, and
function is thus incomplete if it does not take into account its higher-order structure and the
behaviour of its principal units—the chromosomes.
The chromosome theory of heredity, which linked the behaviour of Mendel’s “factors’’
(units of inheritance) with that of chromosomes, was coined by Walter S. Sutton more than a
century ago. This was followed, only a few decades later by Cyril D. Darlington’s demonstra-
tion that the behaviour of chromosomes and meiotic crossing over in particular, was the main
force behind evolution as opposed to single gene mutations and deletions. This set in motion
the quest to understand the nature of inheritance, leading to the discovery of the structure of
DNA and the advent of molecular biology and genomics. At this point the goal seemed clear—
all that was needed was to establish the sequence of bases in the DNA. However, as increasing
amounts of DNA sequences were generated, it became obvious that there was still a lot to
discover about how DNA was organized within chromosomes and how the DNA sequence
information was interpreted, processed, and utilized in the nuclear and cellular environments.
The days when the DNA sequence itself was considered a holy grail are over and we now
know that things are considerably more complicated.
Luckily, progress in genomics has been complemented by advances in understanding the
dynamic structure of chromatin, the organization of interphase nuclei, and the behaviour of
chromosomes during mitosis and meiosis. The latter includes novel insights into modified cell
cycles, which may lead to chromosomes with more than two chromatids. Impressive progress
has also been made in understanding the origin and function of specialized chromosomes (e.g.,
B chromosomes and sex chromosomes) and in appreciating the extent and significance of
polyploidy in plant evolution. Although the frequent occurrence of polyploidy has been known
for a long time based on chromosome counts and behaviour, the advent of DNA sequencing
and comparative genomics has been instrumental in uncovering evidence of further rounds of
polyploidy buried within the genome and now no longer visible at the chromosome level. Such
studies have reinforced and extended our understanding of the significance of this mechanism
as one of the main forces underlying the evolution and large diversity of many plant genomes.
The effect has been multiplied by the extensive structural chromosome changes, which,
together with alterations in chromosome number and genome size, can accompany plant
speciation.

v
vi Preface

To fully understand and appreciate the diversity, functioning, and evolution of plant
genomes, a holistic knowledge of the current status in each of these individual areas is vital,
yet there is no single accessible source of information currently available. Thus, we felt it
timely to fill this gap.
This book is the second volume of a two-volume set on Plant Genome Diversity. Our aim is
to assist students and researchers by providing as complete an overview as possible of each
respective area of research. We have succeeded in engaging leading experts in each field who
describe the current state-of-the-art knowledge without overwhelming the reader with details
that can be found elsewhere. What we offer in the present volume are 20 chapters whose topics
have been chosen carefully to provide a complete picture. Each chapter can stand on its own
and thus the reader does not need to read all chapters if he/she is only interested in a specific
area. We sincerely hope that this model serves our readers well. It is up to them to decide if we
have succeeded.
The 20 chapters deal with individual aspects of plant genome structure, function, and
evolution and they are divided into five informal sections.

Evolutionary Framework for Studying the Diversity of Plant Genomes

Although we do not necessarily expect our readers to read all chapters, we do recommend that
those interested in the evolution of plant genomes read the first chapter by Soltis and Soltis
(Chap. 1) who provide an overview of plant phylogeny, with an emphasis on angiosperms.
Among other things, they highlight research projects that have deposited phylogenetic trees in
public databases and can be downloaded for analysis.

Architecture and Dynamics of the Plant Cell Nucleus

In nondiving cells, the chromosomes are organized within the nucleus, although the structural
and functional complexity of this organization is still poorly understood. One can hardly
imagine the intricacy of interactions of DNA with various molecules necessary to control tens
of thousands of genes and process transcripts of genic and non-genic DNA. In addition, this is
all taking place in a tightly packed nuclear environment, which also harbors structures needed
for DNA synthesis and repair, chromosome reduplication, posttranscriptional modifications,
and synthesis of ribosomal subunits, to name but a few. Jones and Langdon (Chap. 2) review
nuclear organization and discuss the consequences of interspecific hybridization, which results
in two different genomes being accommodated within a single nucleus. This cohabitation may
not be peaceful and can result in dramatic structural and epigenetic reorganizations in
subsequent generations.
The way DNA is organized and packaged into chromatin, particularly at the higher-order
level has never been entirely clear although numerous models have been proposed. However,
recent discoveries question even the existence of the 30-nm fibre, which traditionally has been
considered to originate by folding the 11 nm nucleosome fibre. In Chap. 3, Takata et al.
address this topic by describing the composition of chromatin in relation to chromosome
condensation and DNA packing. Moreover, they present a novel model for chromosome
structure, which suggests that the nucleosome fibres exist in a highly disordered state and do
not form 30-nm chromatin fibres at all.
Preface vii

In addition to separating the nuclear environment from the cytoplasm, the nuclear envelope
performs many important functions; one of which is the control of molecular traffic between
both cellular compartments. Kiseleva et al. (Chap. 4) describe the composition of the nuclear
envelope, the nuclear pore complexes, and their assembly and function and discuss possible
interactions of the envelope with the cytoplasmic and nucleoplasmic components.
Nuclei are known to contain a variety of nuclear bodies, but only the nucleolus can be easily
identified by optical light microcopy. It is where the cell produces ribosomes, which are
required by the cell in large numbers. Shaw (Chap. 5) summarizes the current state of
knowledge of the nucleolus, which is formed on nucleolar organizing regions of chromo-
somes.
For plants to grow and reproduce, the cells must divide either through mitosis or meiosis.
The aim is that the hereditary material is faithfully transmitted to the daughter cells. Not only
must the chromosomes be fully reduplicated but their chromatids must separate at the right
moment and move in the right direction to form daughter nuclei. In addition, the nuclear
envelope breaks down during cell division and this represents an additional major challenge
for the genetic apparatus. Magyar et al. (Chap. 6) provide an insightful review on molecular
events underlying the mitotic cell cycle and mitosis itself.
Following cell division, cell and tissue differentiation is often accompanied by modified
cell cycles in which the mitosis step is omitted and the nuclear envelope does not break down.
Maluszynska et al. (Chap. 7) outline these different types of endopolyploidy and describe the
molecular pathways involved in switching from the mitotic to the endopolyploidization cycle
and how the number of endocycles are regulated. They also review the occurrence of
endopolyploidy, its biological significance, and the structure of endopolyploid nuclei.
The production of gametes provides an important means to generate genetic variation via
recombination of parental chromatids and their random segregation. Given the complexity of
the process, it is not surprising that it is unclear exactly how the mitotic machinery is modified
for the purpose of meiosis. Nevertheless, Jenczewski et al. (Chap. 8) describe the current
knowledge in this area, covering chromosome dynamics during meiosis, initiation of meiotic
recombination, regulation of double strand break repair, crossover formation and interference,
genetic control of crossing-over formation, and its distribution in polyploids.

Karyotype Diversity Across Plants and Trends in Evolution

One of the ways in which the diversity of plant genomes is manifested is through a wide range
of chromosome numbers. Lysák and Schubert (Chap. 9) explain that in many cases this
originates via chromosome rearrangements. The authors outline in detail the mechanisms of
chromosome rearrangements detectable by microscopic techniques and highlight those that
have had an impact on the alteration of chromosome number and structure during evolution
and thus may have played a role in speciation.
The compartmentalization of genomes into chromosomes has provided opportunities for
the development of specialized chromosomes. One such example is the B chromosome (often
called supernumerary chromosome), and Houben et al. (Chap. 10) describe its structure, DNA
composition, and evolution. The authors explain peculiarities in the behaviour of Bs during
mitosis and meiosis and list various drive mechanisms responsible for retaining Bs in the
population.
Sex chromosomes are another classic example of specialized chromosomes, and Janoušek
et al. (Chap. 11) review sex determination systems in various plant groups and, based on
taxonomic distribution, argue that dioecy has originated independently many times during
evolution. The authors introduce the genus Silene as an excellent system to study the evolution
of sex chromosomes and present the first ever evidence of sex dimorphism in dioecious plants.
viii Preface

Bureš et al. (Chap. 12) describe holocentric chromosomes which differ from the more
common monocentric chromosomes by the way in which spindle microtubules attach along
the whole chromosome length through kinetochores that cover a substantial part of their
poleward surfaces during mitosis. They review the occurrence of holocentric chromosomes
in plants and describe their chromatin structure and behaviour during mitosis and meiosis and
the evolutionary processes that have contributed to the diversity of holocentric karyotypes.
The remaining three chapters in this section analyse karyotype diversity in three different
groups of plants. Weiss-Schneeweiss and Schneeweiss (Chap. 13) provide a comprehensive
account of karyotype diversity and evolutionary trends in angiosperms. They discuss in detail
how changes in chromosome number, including dysploidy and aneuploidy, as well as changes
in chromosome morphology contribute to the karyotype diversity observed. They also outline
various cytogenetic methods which can be used to characterize chromosomes in a karyotype
and study their changes during evolution and speciation. Murray (Chap. 14) presents a survey
of chromosome numbers and size variation in gymnosperms, the sister group to angiosperms,
and describes the methods used to analyse karyotype diversity in this group of seed plants.
After reviewing available data, the author concludes that in contrast to angiosperms, gymnos-
perms are characterized by much greater uniformity in chromosome number and karyotype.
The third chapter in this block is by Barker (Chap. 15) who focuses on karyotype and genome
evolution in pteridophytes (monilophytes and lycophytes). He draws attention to the high
chromosome numbers typical of many ferns, particularly the homosporous species, which on
average contain over three-fold more chromosomes than the average flowering plant. Interest-
ingly, there is currently no conclusive answer as to why this should be so although it is
expected that the availability of complete genome sequences will contribute to solving this
long-standing mystery.

Generative Polyploidy

The three chapters in this section evaluate various features of generative polyploidy, which is
widespread in land plants. Husband et al. (Chap. 16) examine patterns of polyploid occur-
rence, such as the variation among taxonomic groups at or above the species level, intraspe-
cific variation, variation in mechanisms of formation, geographic and ecological patterns of
polyploid incidence, and associations between ploidy and reproduction.
Thanks to the advances in DNA sequencing and genomics there is now evidence to suggest
that most seed plants have undergone at least one episode of poylploidization. Thus, one
cannot consider the evolution of land plants without understanding polyploidy. Fawcett et al.
(Chap. 17) explain how the episodes of ancient polyploidization can be identified and dated
and describe the immediate consequences of polyploidization to genes and genomes. They
also discuss changes within polyploid genomes during evolution and the contribution of
polyploidization to the evolutionary success of descendant lineages.
While the majority of studies on polyploidy have focused on angiosperms, Rensing et al.
(Chap. 18) consider the importance of polyploidy in haploid-dominant land plants, the
bryophytes. Here, polyploidy may play an even more essential evolutionary role than in
other evolutionary lineages, rendering a bryophyte more robust against somatic mutations,
while changes in chromosome number through polyploidy can lead to changes in the sexual
system. The need for more genomic data and model species is paramount and the sequencing
of the genome of the moss Physcomitrella patens together with the eagerly anticipated
genome sequences from other moss species and the liverwort Marchantia polymorpha in the
near future should shed further light on genome evolution and the role of polyploidy in these
haploid-dominant land plants.
Preface ix

Genome Size Diversity and Consequences

The book closes with chapters that consider the whole genome in bulk. Leitch and Leitch
(Chap. 19) take advantage of the recent increase in the number of species with genome size
data and provide a comprehensive review on diversity of genome sizes across all groups of
land plants. Evaluation of individual groups suggests that most plant genomes are rather small,
probably due to strong selection pressure to limit genome size. Importantly, the chapter also
considers how the diversity in genome size might have evolved. The last chapter of this
volume by Greilhuber and Leitch (Chap. 20) examines the phenotypic correlates of variation
in genome size, which include cell size and cell division rate. It also discusses the theories to
explain the causality behind this variation observed, considers alternative views, and puts
important studies into focus.

There is no doubt that it was an ambitious goal to cover the broad range of biological
phenomena related to the structure, function, and evolution of plant genomes. However, we
were motivated by the lack of a single resource, which is so needed in this era of rapid DNA
sequence data generation. The chapters included in this volume deliver exciting facts from the
history and life of plant genomes and present unanswered questions and hypotheses. We hope
that the readers will find that the time spent with the book is both enjoyable and stimulating.
This volume would not exist without the contribution of the authors of individual chapters.
Busy leaders in their areas of research, they spared precious time to share with us their
knowledge and visions. We cannot be grateful enough for this and we appreciate their efforts
and patience when responding to our requests for revisions. The only reward for them may be a
response from the readers. So why not contact them? Sincere thanks go to the publisher,
Springer-Verlag, Vienna and New York, who initiated and accompanied this project and made
the publication of this volume possible. We appreciate the careful and professional manage-
ment of the project.

RBG Kew, United Kingdom Ilia. J. Leitch

Vienna, Austria Johann Greilhuber
Olomouc, Czech Republic Jaroslav Doležel
Ames, Iowa, USA Jonathan F. Wendel
March, 2012
.
Contents

1 Angiosperm Phylogeny: A Framework for Studies of Genome Evolution . . . . . . . 1

Pamela S. Soltis and Douglas E. Soltis

2 The Plant Nucleus at War and Peace: Genome Organization in the Interphase
Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
R. Neil Jones and Tim Langdon

3 The Organization of Genomic DNA in Mitotic Chromosomes: A Novel View 33

Hideaki Takata, Sachihiro Matsunaga, and Kazuhiro Maeshima

4 Structural Organization of the Plant Nucleus: Nuclear Envelope, Pore Complexes

and Nucleoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Elena Kiseleva, Jindriska Fiserova, and Martin W. Goldberg

5 The Plant Nucleolus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Peter Shaw

6 Cell Cycle Modules in Plants for Entry into Proliferation and for Mitosis . . . . 77
Zoltán Magyar, Masaki Ito, Pavla Binarová, Binish Mohamed, and Laszlo Bogre

7 Endopolyploidy in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Jolanta Maluszynska, Bozena Kolano, and Hanna Sas-Nowosielska

8 Meiosis: Recombination and the Control of Cell Division . . . . . . . . . . . . . . . . . . . . . . 121

Eric Jenczewski, Raphael Mercier, Nicolas Macaisne, and Christine Mézard

9 Mechanisms of Chromosome Rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Martin A. Lysák and Ingo Schubert

10 Biology and Evolution of B Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Andreas Houben, Ali Mohammad Banaei-Moghaddam, and Sonja Klemme

11 Chromosomes and Sex Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Bohuslav Janoušek, Roman Hobza, and Boris Vyskot

12 Holocentric Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Petr Bureš, František Zedek, and Michaela Marková

13 Karyotype Diversity and Evolutionary Trends in Angiosperms . . . . . . . . . . . . . . . 209

Hanna Weiss-Schneeweiss and Gerald M. Schneeweiss

xi
xii Contents

14 Karyotype Variation and Evolution in Gymnosperms . . . . . . . . . . . . . . . . . . . . . . . 231

Brian G. Murray

15 Karyotype and Genome Evolution in Pteridophytes . . . . . . . . . . . . . . . . . . . . . . . 245

Michael S. Barker

16 The Incidence of Polyploidy in Natural Plant Populations: Major Patterns and

Evolutionary Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Brian C. Husband, Sarah J. Baldwin, and Jan Suda

17 Significance and Biological Consequences of Polyploidization in Land Plant

Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Jeffrey A. Fawcett, Yves Van de Peer, and Steven Maere

18 Evolutionary Importance of Generative Polyploidy for Genome Evolution of

Haploid-Dominant Land Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Stefan A. Rensing, Anna K. Beike, and Daniel Lang

19 Genome Size Diversity and Evolution in Land Plants . . . . . . . . . . . . . . . . . . . . . . . 307

Ilia J. Leitch and Andrew R. Leitch

20 Genome Size and the Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

Johann Greilhuber and Ilia J. Leitch

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Contributors

Dr. Sarah J. Baldwin Department of Integrative Biology, University of Guelph, Guelph,

ON, Canada
Michael S. Barker Department of Ecology & Evolutionary Biology, University of Arizona,
Tucson, USA, msbarker@email.arizona.edu
Dr. Anna K. Beike Plant Biotechnology, Faculty of Biology, University of Freiburg,
Freiburg, Germany
Dr. Pavla Binarová Institute of Microbiology, ASCR, Prague 4, Czech Republic
Prof. Laszlo Bogre Royal Holloway, University of London, Centre for Systems and
Synthetic Biology, Egham, UK, l.bogre@rhul.ac.uk
Prof. RNDr. Petr Bureš Department of Botany and Zoology, Faculty of Science, Masaryk
University, Brno, Czech Republic, bures@sci.muni.cz
Dr. Jeffrey A. Fawcett Graduate University for Advanced Studies, Hayama, Kanagawa, Japan
Dr. Jindriska Fiserova Department of Biological and Biomedical Sciences, Durham
University, Durham, UK
Dr. Martin W. Goldberg Department of Biological and Biomedical Sciences, Durham
University, Durham, UK
Prof. Johann Greilhuber Department of Systematic and Evolutionary Botany, Faculty
of Life Sciences, University of Vienna, Vienna, Austria
Dr. Roman Hobza Institute of Biophysics, Academy of Sciences of the Czech Republic,
Brno, Czech Republic
Dr. Andreas Houben Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Gatersleben, Germany, houben@ipk-gatersleben.de
Prof. Dr. Brian C. Husband Department of Integrative Biology, Science Complex,
University of Guelph, Guelph, ON, Canada, bhusband@uoguelph.ca
Dr. Masaki Ito Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya,
Japan
Dr. Bohuslav Janoušek Institute of Biophysics, Academy of Sciences of the Czech
Republic, Brno, Czech Republic
Dr. Eric Jenczewski Institut Jean-Pierre Bourgin, Institut National de Recherche
Agronomique, cedex, France
Prof. R. Neil Jones Institute of Biological, Environmental and Rural Sciences, Aberystwyth
University, Wales, UK, rnj@aber.ac.uk

xiii
xiv Contributors

Dr. Elena Kiseleva Laboratory of Morphology and Function of Cell Structure, Institute
of Cytology and Genetics, Novosibirsk, Russia, elka@bionet.nsc.ru
Dr. Sonja Klemme Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Chromosome Structure and Function Laboratory, Gatersleben, Germany
Dr. Bozena Kolano Department of Plant Anatomy and Cytology, University of Silesia,
Katowice, Poland
Dr. Daniel Lang Plant Biotechnology, Faculty of Biology, University of Freiburg, Freiburg,
Germany
Dr. Tim Langdon Institute of Biological, Environmental and Rural Sciences, Aberystwyth
University, Wales, UK
Prof. Andrew R. Leitch School of Biological and Chemical Sciences, Queen Mary,
University of London, London, UK
Dr. Ilia J. Leitch Jodrell Laboratory Royal Botanic Gardens, Kew, Richmond, Surrey, UK,
I.Leitch@kew.org
Dr. Martin Lysák Department of Experimental Biology, Faculty of Science, Masaryk
University, Brno, Czech Republic, lysak@sci.muni.cz
Dr. Nicolas Macaisne Institut Jean-Pierre Bourgin, Institut National de Recherche
Agronomique, Versailles, cedex, France
Prof. Steven Maere Department of Plant Systems Biology, VIB, Ghent, Belgium,
stmae@psb.vib-ugent.be
Dr. Kazuhiro Maeshima Laboratory for Biological Macromolecules, Structural Biology
Center, National Institute of Genetics, Mishima, Shizuoka, Japan, kmaeshim@lab.nig.ac.jp
Dr. Zoltán Magyar Institute of Plant Biology, Biological Research Centre, Szeged,
Hungary
Prof. Jolanta Maluszynska Department of Plant Anatomy and Cytology, University
of Silesia, Katowice, Poland, jolanta.maluszynska@us.edu.pl
Dr. Michaela Marková Department of Botany and Zoology, Faculty of Science, Masaryk
University, Brno, Czech Republic
Dr. Sachihiro Matsunaga Department of Biotechnology, Graduate School of Engineering,
Osaka University, Suita, Osaka, Japan
Dr. Raphael Mercier Institut Jean-Pierre Bourgin, Institut National de Recherche
Agronomique, Versailles, cedex, France
Prof. Christine Mézard Station de Génétique et d’Amélioration des Plantes, Versailles,
France, Christine.Mezard@versailles.inra.fr
Dr. Binish Mohamed Royal Holloway, University of London, Centre for Systems
and Synthetic Biology, Egham, UK
Dr. Ali Mohammad Banaei Moghaddam Leibniz Institute of Plant Genetics and Crop
Plant Research (IPK), Gatersleben, Germany
Prof. Brian G. Murray School of Biological Sciences, University of Auckland, Auckland,
New Zealand, b.murray@auckland.ac.nz
Dr. Stefan A. Rensing FRISYS, Faculty of Biology, University of Freiburg, Freiburg,
Germany, stefan.rensing@biologie.uni-freiburg.de
Contributors xv

Prof. Ingo Schubert Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Gatersleben, Germany
Dr. Hanna Sas-Nowosielska Department of Plant Anatomy and Cytology, University of
Silesia, Katowice, Poland
Prof. Gerald M. Schneeweiss Department of Systematic and Evolutionary Botany, Faculty
Center Botany, University of Vienna, Vienna, Austria
Dr. Peter Shaw Cell and Developmental Biology Department, John Innes Centre, Norwich,
UK, peter.shaw@bbsrc.ac.uk
Prof. Douglas E. Soltis Department of Biology and the Genetics Institute, University
of Florida, Gainesville, FL, USA, dsoltis@botany.ufl.edu
Prof. Pamela S. Soltis Laboratory of Molecular Systematics and Evolutionary Genetics,
Florida Museum of Natural History, University of Florida, Gainesville, FL, USA,
psoltis@flmnh.ufl.edu
Prof. Jan Suda Department of Botany, Faculty of Science, Charles University in Prague,
Prague, Czech Republic
Dr. Hideaki Takata Biological Macromolecules Laboratory, Structural Biology Center,
National Institute of Genetics, Mishima, Shizuoka, Japan
Prof. Yves Van de Peer Department of Plant Systems Biology, VIB, Ghent, Belgium, yves.
vandepeer@psb.vib-ugent.be
Prof. Boris Vyskot Institute of Biophysics, Czech Academy of Sciences, Laboratory of
Plant Developmental Genetics, Brno, Czech Republic, vyskot@ibp.cz
Prof. Hanna Weiss-Schneeweiss Department of Systematic and Evolutionary Botany,
Faculty Center Botany, University of Vienna, Vienna, Austria, Hanna.Weiss@univie.ac.at
Dr. František Zedek Department of Botany and Zoology, Faculty of Science, Masaryk
University, Brno, Czech Republic
.
 Angiosperm Phylogeny: A Framework for Studies
of Genome Evolution 1
Pamela S. Soltis and Douglas E. Soltis

Contents 1.1 Introduction

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
The angiosperms—or flowering plants—comprise an
1.2 Methods of Phylogenetic Analysis: A Primer . . . . . . . . . . . . . 2
estimated 260,000 (Takhtajan 1997)–400,000 (Raven in
1.3 The Phylogeny of Embryophytes: An Abbreviated Jarvis 2007) extant species and occupy nearly all habitats
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
on Earth except the coldest arctic and polar regions and the
1.4 The Phylogeny of Angiosperms: An Overview . . . . . . . . . . . 5 deepest oceans. Their diversification has occurred over a
1.4.1 Major Clades . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 relatively short timespan, with the fossil record placing the
1.4.2 Repeated Radiations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.4.3 Unresolved Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 earliest angiosperms in the early Cretaceous, approximately
1.4.4 “Big Trees” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 132 million years ago. Molecular clock estimates suggest
1.5 Studies of Genome Evolution in Angiosperms . . . . . . . . . . . . 7
that the angiosperms are perhaps older, dating to the Jurassic
(e.g., Sanderson et al. 2004; Bell et al. 2005; Bell et al. 2010)
1.6 Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
or even the Triassic (Magallon 2010; Smith et al. 2010).
1.6.1 New Scope, New Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.6.2 Improved Access to Data, Trees, and Tools . . . . . . . . . . . . . . . . . 8 Our understanding of the phylogeny of angiosperms has
improved dramatically in recent years through large-scale
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
collaborative analyses (e.g., Chase et al. 1993; Soltis et al.
1999; Soltis et al. 2000; Hilu et al. 2003; Soltis et al. 2011) and
the application of molecular data, from single genes to entire
plastid genomes (e.g., Jansen et al. 2007; Moore et al. 2007;
Moore et al. 2010). Likewise, many clade-specific analyses
have clarified relationships within some of the largest groups
of angiosperms: e.g., Monocotyledoneae (monocots sensu
Cantino et al. 2007; subsequent italicized names refer
to phylogenetically defined clades in Cantino et al. 2007),
Chase et al. (2006); Caryophyllales, Brockington et al.
(2009); Eudicotyledoneae (eudicots), Moore et al. (2010);
Campanulideae (campanulids), Tank and Donoghue (2010).
In less than 20 years time, our view of angiosperm phylogeny
has been transformed from a nebulous series of possible
transitions to a well-supported and well-resolved tree of
explicit sister-group relationships (summarized in Fig. 1.1).
The stability of this tree is reflected in the modest changes to
the classification of the Angiosperm Phylogeny Group
(APG) over the past decade (1998, 2003, 2009; summarized
by Stevens 2001 onward) and in the development of
a phylogenetic nomenclature for angiosperms (Cantino
P.S. Soltis (*)
Florida Museum of Natural History and the Genetics Institute,
et al. 2007).
University of Florida, Gainesville, FL 32611, USA
e-mail: psoltis@flmnh.ufl.edu

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 1

DOI 10.1007/978-3-7091-1160-4_1, # Springer-Verlag Wien 2013
2 P.S. Soltis and D.E. Soltis
Fig. 1.1 (continued)
Phylogenetic trees of angiosperms have been used to
address a range of evolutionary and ecological questions,
such as the causes of diversification (Davies et al. 2004), the
evolution of reproductive systems (e.g., Culley et al. 2002),
the evolution of syncarpy and its role in pollination
(Armbruster et al. 2002), and the relationship among phy-
logeny, biogeography, and biodiversity (Donoghue 2008). In
addition, trees for which internal nodes have been dated (e.g.,
Wikstrom et al. 2001; Bell et al. 2005; Bell et al. 2010) have
supplied a framework for many additional studies (Slingsby
and Verboom 2006; Vamosi et al. 2006; Edwards et al. 2007;
Webb et al. 2008).
Recent advances in angiosperm phylogenetics have also
played a significant role in selecting taxa for genetic analysis
and genome sequencing (e.g., Pryer et al. 2002; Soltis et al.
2008). For example, studies of gene family evolution have
focused on representatives of basal angiosperm clades, basal
eudicots, and selected monocots and eudicots (e.g., Kim et al.
2004; Kim et al. 2005; Zahn et al. 2005) to investigate patterns
of gene duplication and loss. The results yield complex
patterns of gene family dynamics—patterns that are not
apparent through analysis of model systems alone. Likewise,
genomic resources (e.g., BAC libraries) have been developed
for a set of phylogenetically important plant species in antici-
pation of eventual genomic analysis and sequencing. Most
recently, genome sequencing of Amborella trichopoda, the
sister to all other extant angiosperms (e.g., Soltis et al. 1999;
Soltis et al. 2000; Hilu et al. 2003; Leebens-Mack et al. 2005;
Jansen et al. 2007), has been initiated, to provide an evolu-
tionary reference for genome analysis within the angiosperms
and across all green plants (Soltis et al. 2008; Chamala et al.
2011). The genome sequence of Aquilegia of Ranunculales,
the sister to all other eudicots, will similarly provide an
evolutionary reference for eudicots and a further point of
comparison among the genomes of Amborella, monocots,
and model eudicots.
Here we provide an overview of plant phylogeny, with an
emphasis on angiosperms, based on the past two decades of
research, to serve as the basis for investigating patterns of
genome evolution. We give a summary, as well as many
original citations, with an emphasis on those analyses that
have deposited trees in public databases, such as TreeBASE,
where they are available for download and analysis.
1.2 Methods of Phylogenetic Analysis:
A Primer
The development of phylogenetic methods during the past
decade has produced a perhaps baffling array of approaches,
algorithms, and software. The state of the art a mere decade
ago was maximum parsimony, with numerous options, e.g.,
TNT (Goloboff 1999), parsimony ratchet (Nixon 1999), to
allow for analysis of perhaps several hundred taxa to a few
thousand (Kallersjo et al. 1999) and one or a handful of genes.
Concerns that sufficient tree space was searched were para-
mount, given the restrictions in memory and speed of most
computers at the time. Maximum likelihood analyses were
possible for only tens of taxa. In the early 2000s, major shifts
occurred to model-based approaches as Bayesian methods
(MrBayes, Huelsenbeck and Ronquist 2001; Huelsenbeck
et al. 2001; and then BEAST, Drummond and Rambaut
1 Angiosperm Phylogeny: A Framework for Studies of Genome Evolution 3

b 76
100
Fagales
100
84 Cucurbitales
100 100
Rosales
100
57 Fabales
82 Fabidae
59 Oxalidales
100
99 100 Malpighiales
100 Celastrales
100

Superrosidae
Zygophyllales
100
Malvales
85
100 100
Brassicales
100
100 83 Huerteales
99
Malvidae
100 Sapindales
85 99
Picramniaceae
97 100
Crossosomatales
68
79 Geraniales
100 100
Myrtales
100
Vitaceae
100
Saxifragales
100
Lamiales
100
Boraginaceae

Eudicotyledoneae
73 100

Gunneridae
100
Solanales
100
53
Gentianales
Vahliaceae Lamiidae
100 Oncothecaceae
100

Superasteridae
69 Garryales
Icacinaceae
100
99 100 Asterales
91
Escalloniales
100
100 Apiales
99
Paracryphiales Campanulidae
97 100
100 Dipsacales
100 100
Bruniales
100 Aquifoliales
100
Ericales
100
Cornales
75 100 Berberidopsidales
87
98 100 Caryophyllales
97 100
100 Santalales
100 Dilleniaceae
100
Gunneraceae
100
Buxaceae
100 100
Trochodendraceae
100 Sabiaceae
100 59
100 Proteales
68 100
Ranunculales
86 Ceratophyllaceae
100
Monocotyledoneae
99 100
100
Magnoliales
100 Laurales
100 Magnoliidae
100
85 91100 Canellales
Piperales
100 100
Chloranthaceae
100 Trimeniaceae
83 100 Schisandraceae Austrobaileyales
Austrobaileyaceae
100
100 Nymphaeaceae
Hydatellaceae
Amborellaceae

Fig. 1.1 Summary of phylogenetic relationships among major clades of green plants (Viridiplantae). (a) Overview, based on consensus of many studies.
(b) Overview of angiosperm phylogeny, based on maximum likelihood analysis, with bootstrap values, redrawn from Soltis et al. (2011)

2007), along with maximum likelihood approaches using new
algorithms (e.g., genetic algorithm, GARLI, Zwickl 2006;
RAxML, Stamatakis 2006; Stamatakis et al. 2008), made it
possible to reconstruct large trees (hundreds of taxa) with
confidence scores (posterior probabilities or bootstrap values,
respectively). Parallelization has helped to reduce run times
dramatically for large problems, but has not been universally
implemented to date (although dividing bootstrap analyses
among an array of processors in a cluster is a form of parallel
analysis that can considerably shorten run times).
4 P.S. Soltis and D.E. Soltis
Our assessment is that most projects today employ both
parsimony and at least one model-based approach (typically
RAxML or MrBayes).
Whereas many analyses of phylogeny reconstruction
have embraced model-based methods, most analyses of
character evolution continue to rely on parsimony, despite
the implementation of both maximum likelihood and Bayes-
ian methods for inferring ancestral states and mapping char-
acter variation. Although the reason for this bias is unclear, it
may be that researchers are more comfortable applying
statistical methods to tree selection than to character
mapping, in which parsimony has an intuitive appeal.
Although as in tree selection, likelihood, Bayesian, and
parsimony methods typically produce similar patterns of
character evolution, parsimony results may differ from like-
lihood and Bayesian reconstructions, particularly when
branch lengths are short. We encourage expanded use of
likelihood and/or Bayesian methods for character
reconstructions, at least for comparison with parsimony
results.
Most analyses of plant phylogeny to date have focused on
plastid genes, with an emphasis at deep levels on rbcL, atpB,
ndhF, and to some extent matK. The former two have similar
rates of evolution and are easily alignable, ndhF tends to evolve
slightly more rapidly and is longer (although only part of the
gene is sometimes used), and matK has a higher rate of both
nucleotide substitution and indels, leading to more difficult
alignment. Of course, plastid genes provide only the evolution-
ary history of the plastid, and although this may not be a
concern at the deepest levels of plant phylogeny, one must
consider how well a plastid gene tree may reflect the organis-
mal tree. To date, at deep levels, the only nuclear genes that
have been used widely are the 18S and 26S ribosomal RNA
genes. A number of MADS-box genes have been shown to
track angiosperm phylogeny (Litt and Irish 2003; Kim et al.
2004; Kim et al. 2005; Zahn et al. 2005), but these genes have
not yet been applied solely for the purpose of phylogeny
reconstrution. However, a number of other nuclear genes (or
their introns) have been used at more shallow levels: LEAFY,
APETALA3, PISTILLATA, ALCOHOL DEHYDROGENASE,
GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE,
CHALCONE SYNTHASE, and WAXY, to name a few. All of
this latter set of genes tend to have regions of 1,000 bp (plus or
minus a few hundred) and are generally fairly easy to amplify
with standard primers. However, the use of nuclear genes
carries its own concerns, most notably issues of orthology,
allelic diversity, and recombination, often requiring extensive
cloning, sequencing of clones, and analyses of recombination
prior to phylogenetic analysis. A set of mitochondrial genes
(e.g., matR, atp1, nad5, rps3) has also been applied to plant
phylogeny. These genes tend to evolve more slowly than either
plastid or nuclear genes used to date and can supply characters
that are useful deep in plant phylogeny. However,
mitochondrial-based trees have shown evidence of horizontal
transfer of mitochondrial genes (e.g., Won and Renner 2003;
Bergthorsson et al. 2004; Davis and Wurdack 2004) and should
therefore be used in conjunction with other markers, particu-
larly in groups that contain parasites.
1.3 The Phylogeny of Embryophytes:
An Abbreviated Overview
A thorough summary of plant phylogeny is beyond the scope
of this chapter; instead, we present a simple overview to set
the stage for further discussion of genome evolution in plants
and the phylogenetic placement of angiosperms, the focus of
this chapter. Although important for understanding major
patterns of plant evolution, especially in morphological and
anatomical characters, the fossil record plays a less crucial
role in understanding genome evolution, and we have there-
fore largely confined our discussion of plant phylogeny to
extant taxa. However, the fossil record provides a requisite
timeframe on our interpretation of phylogeny, and the phy-
logenetic placement of fossil groups may affect the topology
of extant groups; we therefore introduce data from fossils as
needed in this brief overview but we recognize that our
treatment is incomplete.
The “green plants” (sometimes referred to as
Viridiplantae or viridophytes) are a clade of at least half a
million species with a fossil record that extends back nearly
one billion years. They share a common cyanobacterial
endosymbiotic event with red algae and glaucophytes and
can be diagnosed by chlorophyll b, starch as the storage
product for photosynthesis, and a stellate flagellar structure
(e.g., Judd et al. 2008). A basal split in the green plants
produced two clades, the chlorophytes (mostly marine
“green algae”) and streptophytes (which include freshwater
“green algae” and embryophytes). Mesostigma, a freshwater
“alga”, has been identified as the sister to all other
streptophytes. Subsequently branching lineages include the
Klebsormidiales, the Zygnematales, and the Coleochaetales
and Charales, the limits of which are not completely clear.
Chara (and relatives) and Coleochaetales seem to be the
sister group(s) of the embryophytes, although some recent
studies place Zygnematales in this position (e.g., Timme
et al. 2012). Embryophytes, or land plants, trace their history
to at least the Ordovician and began to diversify extensively
in the Silurian and Devonian. Morphological and anatomical
synapomorphies of the embryophytes are a multicellular
sporangium, thick-walled spores, multicellular gametangia,
an embryo, and a cuticle.
Within the embryophytes, the phylogeny of the major
clades is not fully resolved (Fig. 1.1a). For example,
although traditionally recognized as a single taxonomic
group, the bryophytes (consisting of mosses, liverworts,
1 Angiosperm Phylogeny: A Framework for Studies of Genome Evolution
and hornworts) are paraphyletic, and the branching order of
these clades relative to the tracheophytes is not yet clear. All
possible branching orders have been proposed. Most mor-
phological and molecular data support liverworts as sister to
all other embryophytes, and the major remaining disagree-
ment is between the topology of (liverworts, (hornworts,
(mosses + tracheophytes))), which is supported by the
shared feature of a sporophyte apical meristem in mosses
and tracheophytes, and (liverworts, (mosses, (hornworts +
tracheophytes))), which is supported by the persistently
green sporophyte in hornworts and tracheophytes (reviewed
in Judd et al. 2008).
The tracheophytes (Tracheophyta) comprise two major
clades, lycophytes (Lycopodiophyta) and euphyllophytes
(Euphyllophyta). Lycopodiophyta comprises Isoetes, Selagi-
nella, and Lycopodiaceae and forms a clade with some of the
most prominent early vascular plants: Cooksonia,
zosterophytes, and Lepidodendrales (Judd et al. 2008).
Extant Euphyllophyta is united by a plastid genome inver-
sion, multicellular sperm, overtopping, and terminal
sporangia on lateral branches. The clade contains two
major clades, Monilophyta and Lignophyta. The former is
composed of Psilotales, Ophioglossales, Equisetales,
Marattiales, and the leptosporangiate ferns (Leptospor-
angiatae). This assemblage of monilophytes was recognized
by Kenrick and Crane (1997) on the basis of stem anatomy
and was later supported by molecular data as well (Pryer
et al. 2001; Pryer et al. 2004). The Lignophyta comprises
several fossil lineages and the Spermatophyta, the seed
plants.
Relationships among seed plants perhaps remain the most
challenging in plant phylogeny. Extensive extinction within
this clade has undoubtedly contributed to the difficulty of
phylogeny reconstruction. The “gymnosperms” as typically
recognized are paraphyletic and include several clades with
extant members (cycads, Ginkgo, conifers, and gnetophytes)
and several other groups that are only found in the fossil record
(Medullosa, seed ferns, glossopterids, Caytonia, Bennetittales).
However, the paraphyly of the “gymnosperms” is not apparent
in molecular-based trees that typically (but not always) recover
reciprocally monophyletic gymnosperms and angiosperms.
When fossils are included in phylogenetic analyses of seed
plants, disagreements exist with regard to both the placement
of many of the non-flowering seed plants and in the sister group
of the angiosperms, and there is little consensus on the overall
phylogeny of all seed plants. It appears that glossopterids,
Caytonia, and Bennetittales are more closely related to
angiosperms than to other “gymnosperms” but beyond that,
there is little resolution. When only extant seed plants are
considered, disagreement still abounds. Most recent studies
have found a topology in which extant gymnosperms and
angiosperms are sister groups, with cycads either sister to all
other extant gymnosperms or sister to Ginkgo, and with
5
conifers and gnetophytes either sister to each other, or more
often, gnetophytes nested within conifers or within Pinaceae.
Despite extensive study using many sources of data and modes
of analysis, the phylogenetic relationships among extant seed
plants remain unresolved. For analyses that seek to examine
patterns of evolution among angiosperms, this frustrating result
precludes identification of the appropriate outgroup for com-
parative studies.
1.4 The Phylogeny of Angiosperms:
An Overview
1.4.1 Major Clades
Angiosperm phylogeny has been studied extensively in
recent decades, from the perspective of deep-level branching
patterns to clades of closely related species. Ultimately and
ideally, these results will be linked, either through supertree
methods that combine published trees via shared taxa or
through new supermatrix analyses that combine all data
into a single matrix for analysis (see below). Here we will
provide only an overview of the major clades and their
interrelationships, followed by further discussion on some
of the emergent patterns from analyses conducted to date.
Nearly all molecular-based analyses of the past decade
have identified Amborella as the sister to all other extant
angiosperms, most often alone or occasionally with
Nymphaeales (Fig. 1.1b). All analyses are consistent in then
placing Austrobaileyales (comprising Austobaileya, Trimenia,
Illicium, and Schisandraceae) as the sister group to all
other extant angiosperms. This large remainder, the
Mesangiospermae, comprises Magnoliidae + Chloranth-
aceae as sister to Monocotyledoneae + Eudicotyledoneae +
Ceratophyllum (Moore et al. 2007). Although long recognized
as an ancient group, the placement of Chloranthaceae has been
elusive, but recent analyses place them as sister to
Magnoliidae. The relationship among magnoliids,
monocots, and eudicots has been very difficult to disentan-
gle, but plastid genome sequences support the sister-group
relationship of monocots and eudicots (+ Ceratophyllum).
Relationships among major clades of monocots are now
clear, but they do not follow traditional taxonomic
circumscriptions. Acorales are sister to all other extant
monocots, and a grade that includes Alismatales, followed
by Petrosaviaceae, subtends a clade comprising the majority
of monocot species diversity. Pandanales + Dioscoreales are
sister to a clade of (Liliales, (Asparagales + Commelinidae)).
One of the most substantial reorganizations of monocot
classification is based on new understanding of relationships
of the former Liliaceae. Although dismantling of this
large family was proposed many years ago, the placements
of its components have not always been clear. Progress has
6 P.S. Soltis and D.E. Soltis
been substantial, but questions remain. Likewise, relationships
within Asparagales have been difficult to resolve, and
although recent analyses have done much to resolve phylog-
eny, few morphological characters have been idenitifed to
diagnose the component clades. The Commelinidae are
diagnosed by starchy pollen, UV-fluorescent ferulic and
coumaric acids in the cell walls, and Strelitzia-type epicuticu-
lar wax. The clade is large, comprising over 25,000 species,
with diversity spanning grasses to palms. Component clades
are Commelinales, Zingiberales, Arecales, and Poales
(+ Dasypogonaceae).
Within eudicots, a basal grade consisting of Ranunculales,
Proteales, Sabiales, Trochodendrales, and Buxales subtends the
“core eudicots”, or Gunneridae. Gunnerales are sister to the
remaining Gunneridae, the Pentapetalae, which fall into
two major clades (Moore et al. 2010; Soltis et al. 2011):
Superrosidae and Superasteridae (sensu Soltis et al.
2011). Superrosidae comprises Saxifragales, Vitaceae, and
Rosidae, whereas Superasteridae contains Santalales,
Berberidopsidales, Caryophyllales, and Asteridae.
Dilleniaceae, which has been associated with Caryophyllales
in some previous analyses and unplaced in many others,
remains unplaced, with alternative placements in Superrosidae
and Superasteridae (see below). The positions of most major
clades of core eudicots (Gunneridae) remain unchanged rela-
tive to earlier studies (e.g., Soltis et al. 1999; Soltis et al. 2000;
Soltis et al. 2005), although additional resolution has been
obtained in both the Rosidae and Asteridae (see Soltis et al.
2011, and references therein).
Phylogenetic analyses of the angiosperms at these deep
levels have resulted in new classifications, such as the
Angiosperm Phylogeny Group’s (APG) (1998, 2003, 2009)
classifications at the familial and ordinal levels, with rank-
free names assigned to clades corresponding to groups larger
than recognized orders. An alternative rank-free classifica-
tion has also emerged (Cantino et al. 2007), with phyloge-
netic definitions provided for many of the clades that
correspond to those recognized at the ordinal level and
above in the APG system.
1.4.2 Repeated Radiations
A prominent pattern apparent in the angiosperm phyloge-
netic trees is a series of polytomies interspersed by regions
of well-resolved relationships (see Soltis et al. 2005; Soltis
et al. 2008; Wang et al. 2009; Soltis et al. 2010). Although
polytomies may be due to insufficient data or taxon sam-
pling, they may also represent real radiations. Within the
angiosperms, several apparent radiations have persisted
through the addition of new data and more taxa, suggesting
that in fact these radiations are real.
The origin of the angiosperms themselves has often been
considered a rapid radiation, based on the fossil record and
Darwin’s words themselves: “The rapid rise and early
diversification of angiosperms is an abominable mystery. . .”
(Darwin 1903). However, phylogenetic reconstructions sug-
gest instead that the angiosperms radiated, not immediately
upon their origin, but a few nodes subsequent to the common
ancestor of all extant angiosperms (Mathews and Donoghue
1999; Soltis et al. 1999; Soltis et al. 2005). This radiation
corresponds to the diversification of the Mesangiospermae
(sensu Cantino et al. 2007), the clade comprising
magnoliids, Chloranthaceae, monocots, Ceratophyllaceae,
and eudicots—in other words, all angiosperms except
Amborella, Nymphaeales, and Austrobaileyales (see Moore
et al. 2007). Subsequent radiations appear to follow the
origin and early diversification of many large clades of
angiosperms: for example, within the eudicots (Eudicoty-
ledoneae), within the core eudicots (Gunneridae), within
Rosidae (and within the fabid and malvid clades, Fabidae
and Malvidae, of Rosidae), within the Asteridae, and within
clades of monocots, to name a few.
Attempts to find causes for these apparent radiations have
met with mixed success. One of the most extensive analyses
of possible factors associated with radiations addressed both
the early radiation of the angiosperms themselves and
subsequent radiations (Davies et al. 2004). Davies et al.
(2004) tested a range of traits reflecting prominent
hypotheses for the “success” of the angiosperms on rates of
diversification and found no significant association at any
level of the tree. Despite the lack of significance of specific
features, radiations within the angiosperms may be
explained by biotic factors and cospeciation with other
clades. For example, modern ferns diversified alongside
angiosperms, suggesting either that angiosperms provided
new habitats for ferns or that the same causal factors allowed
diversification of both clades (Schneider et al. 2004). More-
over, within the angiosperms, the radiation of the rosids is
associated with radiations in several other clades, such as
ants, amphibians, and even primates (see Wang et al. 2009,
for review). Other possible biotic interactions, such as those
with mycorrhizal fungi, may also have contributed to
radiations of angiosperms.
Recent observations of gene duplications at or near nodes
associated with radiations are suggestive of a causal role of
genetic or genomic factors in these radiations themselves.
For example, coincident gene duplications in multiple
subfamilies of the MADS-box gene family prior to the origin
of the angiosperms raised hypotheses about the role that
these duplications in genes important in the specification of
floral organ identity and other features of the flower may
have played in the early evolution of angiosperms (Buzgo
et al. 2005; De Bodt et al. 2005; Zahn et al. 2005). Likewise,
similar patterns of duplication appear to be associated with
1 Angiosperm Phylogeny: A Framework for Studies of Genome Evolution
the early evolution of the eudicots, again suggesting a causal
role in the floral changes that occurred at that point in
angiosperm phylogeny and a further role in diversification
(for reviews see Soltis et al. 2006; Soltis et al. 2009b).
Finally, duplications in the CYCLOIDEA gene family sug-
gest possible roles in both floral and species diversification
in asterids (Howarth and Donoghue 2006).
Coincident gene duplications at specific nodes are sug-
gestive of whole-genome duplications (WGD; see Buzgo
et al. 2005; De Bodt et al. 2005; Zahn et al. 2005; reviewed
in Soltis et al. 2009a; Soltis et al. 2009b), and it may be that
WGD rather than duplications of specific floral genes trig-
gered radiation. Whole-genome duplication (polyploidy)
has, in fact, been suggested as the impetus for angiosperm
success following the K-T boundary (Fawcett et al. 2009).
Genomic data have revealed unsuspected episodes of WGD
throughout green plants (see below; Blanc and Wolfe 2004;
Cui et al. 2006; Soltis et al. 2009a; Jiao et al. 2011 for
review). In several instances, the clade marked by WGD is
more species-rich than the sister clade that lacks the dupli-
cation; however, genomic data are lacking for a sufficient
number of species to allow for thorough statistical analyses
of heterogeneity of diversification rates (Soltis et al. 2009a).
Additional data for more species, so that WGD events can be
plotted more accurately on a phylogenetic tree, are needed.
1.4.3 Unresolved Relationships
Given the recent progress in angiosperm phylogenetics, few
major issues of deep-level relationships remain, although
problems abound within clades recognized as “orders” and
“families” sensu APG. Among deep-level problems, one of
the most perplexing placements is that of Dilleniaceae,
which occupies different positions depending on the data
set and analysis, from sister to Superrosidae (sensu Soltis
et al. 2011), to sister to Superasteridae (sensu Soltis et al.
2011), to sister to Superasteridae + Superrosidae (see Soltis
et al. 2011, for discussion). Other prominent areas requiring
further analysis include: (1) the branching order among basal
eudicots; (2) relationships among major clades of rosids; (3)
relationships within Malpighiales; (4) Lamiales. Most of
these regions can most likely be resolved with additional
taxa and DNA sequence data, although problem areas such
as Malpighiales have recently received substantial attention,
with at least some progress (Wurdack and Davis 2009).
1.4.4 “Big Trees”
Until recently, most tree reconstruction algorithms could not
handle data sets of 1,000 or more terminals. However, recent
modifications have yielded trees with many thousands of
7
species (e.g., Goloboff et al. 2009; Smith et al. 2009;
Smith et al. 2011). The most recent tree generated by
Smith et al. (2011)—with 55,000 taxa!—used a newly
adapted version of RAxML, demonstrating the ability to
use model-based approaches for “big tree” reconstruction.
The concern with such large trees, however, is their accu-
racy: with so many terminals, the thoroughness of the
searches is reduced, raising the question of the accuracy of
the results. The overall structure of the Smith et al. (2011)
55,000-taxon tree is quite similar to trees based on far fewer
taxa (such as the 640-taxon tree of Soltis et al. 2011),
suggesting that for many purposes, this very large tree will
be very useful. However, without further diagnostics on the
performance of tree reconstruction at this large scale, many
close relationships may require cautious acceptance. And
this may be an issue for studies aimed at reconstructing the
evolution of genes or specific genomic traits at a fine scale.
Nevertheless, breakthroughs in tree reconstruction will
undoubtedly lead to new and exciting opportunities for
learning about the evolution of plant genomes.
An alternative to the supermatrix approach described
above is the construction of a supertree from smaller trees
with overlapping taxa (see Sanderson et al. 1998; Davies
et al. 2004). Supertree methods take advantage of vast
amounts of data collected and analysed in the past to produce
a summary of phylogenetic inferences contained in
published trees. Although not without their own problems,
supertrees offer a solution to generating large phylogenies.
Recent methods that combine elements of supermatrix and
supertree approaches show particular promise.
1.5 Studies of Genome Evolution
in Angiosperms
Hypotheses on patterns of chromosomal evolution in the
angiosperms abound. Classical perspectives were based on
integrated inferences on ancient and recent polyploidy, pro-
cesses of chromosomal fission and fusion, and relative
“advancement” of a taxonomic group. More recently, it has
been possible to test hypotheses of increases and decreases in
chromosome number and genome size by mapping these
characteristics across an explicit phylogenetic tree.
Longstanding hypotheses of chromosomal evolution have
suggested that the ancestral chromosome number for
angiosperms ranged from x ¼ 6–9, with x ¼ 7 a commonly
proposed base number (e.g., Stebbins 1950; Ehrendorfer et al.
1968; Stebbins 1971; Raven 1975; Grant 1981). Chromosome
numbers in angiosperms vary dramatically, from 2n ¼ 4
(e.g., Haplopappus gracilis, Asteraceae) to 2n ¼ c. 640
(Sedum suaveolens, Crassulaceae), a 160-fold difference
(Uhl 1978). However, it is clear that genome size varies
independently of chromosome number, with genome size
8 P.S. Soltis and D.E. Soltis
ranging from 1C ¼ 0.065 pg to 1C ¼ 152.23 pg, a c. 2,400-
fold difference (Greilhuber et al. 2006; Pellicer et al. 2010;
Leitch and Leitch, 2013 this volume). Previous reconstructions
of genome size across angiosperms found that the ancestral
genome was “very small”, with 1C  1.4 pg (Leitch et al.
1998; Soltis et al. 2003), with multiple increases and decreases
in genome size from this ancestral condition.
Another clear attribute of angiosperm genomes is poly-
ploidy, and events of whole-genome duplication have also
been mapped across an angiosperm tree (Soltis et al. 2009a).
Whereas polyploidy has long been recognized as an impor-
tant speciation mechanism in angiosperms, events of
genome duplication were long considered to be confined to
the tips of the tree, with a few putative cases of ancient
polyploidy, e.g., Magnoliaceae, Lauraceae, Salicaceae
(Stebbins 1950; Stebbins 1971). Remarkably, genome
sequencing studies have revealed multiple rounds of genome
duplication throughout the evolutionary history of
angiosperms. The surprising finding that the very small
genome of Arabidopsis thaliana has undergone multiple
rounds of duplication set the stage for investigations of
other unsuspected events of genome duplication, and all
other angiosperms sequenced to date likewise exhibit
signatures of ancient duplication (see Soltis et al. 2009a;
Fawcett et al. 2013, this volume). As more genome
sequences are generated, it will be possible to map these
duplication events more clearly onto a phylogenetic tree.
From there, hypotheses of possible causal effects of genome
duplication may be tested.
1.6 Future Prospects
1.6.1 New Scope, New Tools
Despite the progress in reconstructing the major
relationships within the angiosperms, much remains to be
done to produce a comprehensive phylogeny. One approach
to increasing taxon sampling is to include all species that are
represented in GenBank, regardless of gene. Methods such
as those explored by Driskell et al. (2004) have proven that
even very sparse matrices can yield reasonable phylogenetic
trees. A modification of this approach that includes only
species for which any of a small, specified set of genes has
been sequenced (Smith et al. 2009; Smith et al. 2011) has
resulted in a 55,000-taxon tree, generated using maximum
likelihood (via RAxML, Stamatakis 2006), that has been
used to address the evolution of various traits (Smith et al.
2011). The ability to generate trees of this size is a recent
breakthrough and sets the stage for future large-scale
analyses. Whereas until recently, the limitation in
phylogenetics was computational power, the bottleneck
very soon will be a lack of data. Tools being developed by
the iPlant Collaborative (iplantcollaborative.org) will soon
be available for large-scale tree reconstruction and post-tree
analyses.
An alternative approach to using data fortuitously avail-
able from GenBank is the deliberate generation of new data
for taxa not represented in GenBank. For example, only a
fraction of the estimated 15,000 genera of angiosperms are
included in GenBank. Although most genera are likely not
monophyletic (Judd et al. 2008), generic-level classification
reflects a loose assessment of diversity and therefore a
framework for ongoing phylogenetic analysis. However,
substantial specimen collecting, including samples for
DNA analysis, is needed to conduct such a study. Thus,
given new developments in phylogenetic software, perhaps
the major limitation to a comprehensive angiosperm phylo-
genetic tree is the lack of material for molecular analysis.
1.6.2 Improved Access to Data, Trees,
and Tools
Until now, most phylogenetic reconstructions of morpholog-
ical, physiological, ecological, or other characters have
involved, initially, sharing of trees and data sets by
systematists and, more recently, downloading published
trees and data from public databases such as TreeBASE
(treebase.org) and Dryad (datadryad.org). These analyses
have been necessarily limited to those taxa included in
prior phylogenetic trees, without representation of those
taxa that might be of greater interest from the perspective
of morphology or other traits. A solution is to reconstruct a
new tree that includes such taxa, but large phylogenetic
analyses may not be feasible for those interested in
reconstructing patterns of character evolution. New
approaches, such as an automatically generated tree with
each new GenBank release (every 2 months) and the avail-
ability of data matrices and computational resources for tree
estimation, are being developed by the iPlant Collaborative.
Implementation of these tools will facilitate customized
phylogenetic analyses and lead to “democratization” of
angiosperm phylogenetics. Continued investment in phylo-
genetic cyberinfrastructure to address such issues as reticu-
lation, horizontal gene transfer, and patterns of character
evolution will lead to further advancements in our under-
standing of angiosperm evolution.
Acknowledgments This work was supported in part by the US
National Science Foundation (grants EF-0431266 and PGR-0638595)
and the NSF-funded iPlant Collaborative.
1 Angiosperm Phylogeny: A Framework for Studies of Genome Evolution
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 The Plant Nucleus at War and Peace: Genome
Organization in the Interphase Nucleus 2
R. Neil Jones and Tim Langdon

Contents 2.1 Introduction

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
The organization of the plant nucleus has been seen as a key to
2.2 General Aspects of Organization in the Peaceful
Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 understanding the workings of plants themselves for the past
2.2.1 Chromosome Territories (CT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 two centuries. This aspiration seems to be being finally realised
2.2.2 Modifications of Chromatin and DNA . . . . . . . . . . . . . . . . . . . . . 15 as new sequencing technologies are helping to draw together
2.3 The Nucleus Under Strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 old strands of research as well as supporting novel approaches
2.3.1 Plasticity of Gene Family Organization . . . . . . . . . . . . . . . . . . . . 17 to bridge the gap between cytological and molecular scales of
2.3.2 Limits to Expression Neighbourhoods . . . . . . . . . . . . . . . . . . . . . 18 description; and recent advances in understanding epigenetic
2.3.3 Hybrid Vigour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3.4 Mobile Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
processes and mechanisms are providing a fresh perspective on
studies of interphase organization. The inevitable caveat is that
2.4 The Nucleus at War . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 new questions may need to be answered, as the comparative
2.4.1 Ploidy Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.4.2 Changes at the Gene Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 detail now available throws up additional complexities which
2.4.3 Adjustments at the Chromosome Level . . . . . . . . . . . . . . . . . . . . 23 were previously hidden. Chief among these is the extent to
2.5 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
which the genome is not constant within a species. Limited
surveys of genes and genomic regions in maize revealed some
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
years ago that there was more genetic diversity within this one
species than between humans and chimpanzees; and a more
recent genome wide survey indicates that if anything this was
an underestimate (Gore et al. 2009). More generally in both
animals and plants, intragenic copy number variation in non-
repetitive sequences is being found to rival that seen in hetero-
chromatic repeats; 10% or more of coding sequences show
copy number variation in maize and rice (Ding et al. 2007;
Springer et al. 2009). Nuclear organization in many plants must
therefore be capable of accommodating a wide range of struc-
tural and nucleotide polymorphisms without suffering detri-
mental effects; indeed, maize itself demonstrates that the
hybridization of divergent parents may be strongly advanta-
geous (Shull 1948). This plasticity may explain the ability of
many wide crosses to eventually generate stable derivatives,
which in turn is likely to underpin many of the numerous
examples of reticulate evolution being found. Nevertheless,
interspecific hybridizations have often been shown to trigger
genome wide reactions, frequently followed by structural and
R.N. Jones (*) epigenetic reorganization in subsequent generations, which can
Institute of Biological, Environmental and Rural Sciences,
Aberystwyth University, Edward Llwyd Building, Penglais Campus,
be seen as a war between parental genomes. Here we review
Aberystwyth, Wales SY23 3DA, UK some of the aspects of nuclear organization that may underlie
e-mail: rnj@aber.ac.uk

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 13

DOI 10.1007/978-3-7091-1160-4_2, # Springer-Verlag Wien 2013
14
Fig. 2.1 Highly schematic representation of the disposition of
chromosomes in species with a small (a) (Arabidopsis) and a large
(b) (hexaploid wheat) genome. Probes identify the centromeres (red)
and telomeres (green). The chromosome territories in Arabidopsis are
visualized with chromosome-specific probes from pools of BAC
incompatibilities, and indicate some of the changes that may be
required to restore if not peace, then at least a truce.
2.2 General Aspects of Organization
in the Peaceful Nucleus
A number of significant discoveries on the structure of the
nucleus were made early on, at the limits of what could
then be observed with the light microscope. In 1885 Rabl
first described the arrangement of the chromosomes of
salamanders in terms of the orientation of their centromeres
and telomeres. He explained how the anaphase configuration,
with the telomeres at the nuclear periphery of adjacent daugh-
ter cells and the centromeres at the relic poles, was maintained
through to the following interphase (Rabl 1885). Laibach
(1907) later made the remarkable finding that the number of
chromocentres in Arabidopsis corresponded to the number of
chromosomes, and Heitz (1928) demonstrated the continuity
of the structure of chromosomes throughout the cell cycle in
Pellia endiviifolia, using blocks of constitutive heterochroma-
tin as markers for the identity of individual chromosomes. An
early inference was that the interphase nucleus would be
made up of discrete chromosomal territories (CTs) (Boveri
1909), and each subsequent development of microscopic
techniques has improved our understanding of the nuclear
architecture of CTs and its impact on gene expression
(reviewed in Rouquette et al. 2010). Most recently, direct
3D analysis of living cells using green fluorescent protein
(GFP) tags and BACs, and of accurately fixed material by
R.N. Jones and T. Langdon
contigs; whereas in wheat individual chromosomes, present as a
wheat/rye addition line 1R, can be seen by probing with whole genome
DNA which discriminates between the dispersed repeats of wheat and
rye chromatin
confocal methods, has been extended by indirect analysis via
sequencing of DNA retrieved from common pools of cross
linked proteins (3C, 4C, 5C and HiC methods), which is
providing resolution beyond the limits of microscopy
(Rajapakse and Groudine 2011). Nevertheless, the role and
even the extent of CTs remains unclear, and it appears that
this is as much because of inherent variability as because of
technical limitations.
This variability is well illustrated by the inconsistent
occurrence even of Rabl arrangements. They do appear to
be a fixture of plant species with relatively large genomes
(such as wheat, oat, barley, Vicia faba and Allium cepa, all
with genomes in excess of 1C ¼ 5,000 Mb), where probes
identifying alien segments can be used to show chromosome
arms assuming a string-like form running between the
centromeric and telomeric nuclear poles (Abranches et al.
1998) (Fig. 2.1). On the other hand, plant species with small
genomes (such as sorghum, rice, Arabidopsis, and brassicas,
with genomes of less than 1C ¼ 1,000 Mb) generally lack
the Rabl arrangement. This can be observed, for example,
by 3D reconstruction of living cells of A. thaliana after
tagging with a GFP probe, where the centromeres are pre-
dominantly dispersed around the nuclear periphery in dif-
ferent cell types (Fang and Spector 2005) although the
telomeres are associated within the nucleus core around
the nucleolus (Armstrong et al. 2001). As a result individual
Arabidopsis chromosomes appear as radial euchromatic
loops emanating from the heterochromatic centromeric
chromocentres when painted with specific BAC markers
(Fransz et al. 2002). Maize, with 1C ¼ 3,000 Mb of
2 The Plant Nucleus at War and Peace: Genome Organization in the Interphase Nucleus
DNA, is intermediate in size, and cannot be classified as
either having a Rabl arrangement or not (Cowan et al. 2001)
although rice (1C ¼ 430 Mb) does display a tissue-specific
Rabl arrangement in endopolyploid differentiating cells in the
xylem (Santos and Shaw 2004). Dong and Jiang (1998)
suggested that chromosome, rather than genome, size might
be a crucial factor; and Fransz and de Jong (2011) have
suggested 500 Mb as a threshold value. However, while it
might be reasoned that the larger the chromosome, the greater
the logistical problem of interphase chromatin organization,
and hence the longer persistence of anaphase organization
(Cowan et al. 2001), this is not the full answer, as yeasts may
show a Rabl configuration while larger genome mammals do
not (discussed further in Schubert and Shaw 2011).
2.2.1 Chromosome Territories (CT)
The enigmatic nature of Rabl arrangements also applies to
lower levels of nuclear organization, despite extraordinarily
detailed analyses of interphase CTs in a variety of animal
and plant cell types. As with Rabl, there are examples of
highly ordered structures, which logic suggests must play an
important functional role; however, again as with Rabl, there
are examples where these structures do not occur or are
disrupted without apparent ill effect. These conflicting
observations are described in a number of excellent reviews
(Cremer and Cremer 2010; Woodcock and Ghosh 2010) and
will not be repeated in detail here. At present studies on
human cells support a model of regions of increased gene
expression (ridges) and similar sized domains enriched for
genes with low expression (anti-ridges). Originally defined
by mapping transcriptome data onto linear chromosome
maps, which found one to two orders of magnitude differ-
ence in expression levels between ridges and anti-ridges
(Caron et al. 2001), the patterns have been visualized in
three dimensions using confocal microscopy, which found
anti-ridges to be more compact and regularly organized than
ridges, and to be closer to the nuclear envelope, where they
may be anchored (Goetze et al. 2007). Large cell-to-cell
variation in this organization was seen and interpreted as
structural plasticity rather than experimental error. This is
consistent with global patterns of chromatin organization
revealed indirectly by the HiC procedure, which supports a
fractal globule model where self-organizing domains ‘crum-
ple’ in such a way that the resulting structures do not pene-
trate into other crumples (Lieberman-Aiden et al. 2009).
Accordingly, there are no knots in the structures to prevent
dynamic looping out, whether by diffusion or in response to
specific cues, but defined CTs are expected to persist over
the period of the cell cycle as crumples reform. The HiC data
support a division into two spatial sub-compartments,
15
similar to the ridge/anti-ridge model, and indicate frequent
interchromosomal contact (Lieberman-Aiden et al. 2009).
Studies on plant CTs are not yet as detailed as for humans
but do not conflict with the fractal globule model. Significant
patterns of co-expressed intrachromosomal neighbourhoods
have been found (Williams and Bowles 2004; Riley et al.
2007) and it is likely that chromosome conformation capture
methods will soon allow these to be examined in three
dimensions. Clear demonstrations have been made, however,
that CT orthologues do not co-localize at high frequency
during interphase (reviewed in Schubert and Shaw 2011)
and transcription appears to occur at sites distributed
throughout the nucleus, rather than concentrated in regions
expected to be gene-rich on the basis of, for example, Rabl
disposition. Strong interchromosomal associations may
normally be limited to heterochromatic chromocentres and
nucleolar organizers. Reorganization of chromatin in
response to a number of cues has been followed, including
large scale reversible decondensation of heterochromatin in
Arabidopsis (Tessadori et al. 2007). Reassembly of the
Arabidopsis chromocentres is sequential with an order
which appears to be determined simply by weak interactions
whose strength is proportional to array length. Similarly,
treatment of wheat seedlings with chemicals which disrupt
chromatin silencing, at levels too low to significantly reduce
growth, was found to cause decondensation of chromosome
arms and remodelling of CTs, but did not alter the Rabl
organization of heterochromatic blocks (Santos et al. 2002).
The disposition of chromosome pairs at interphase in
Arabidopsis has been studied by chromosome painting
with pools of chromosome-specific BAC contigs, and it
appears that association patterns on homologous and heter-
ologous chromosomes is largely random, and that only
chromosomes bearing nucleolus organizing regions
(NORs) show that homologs associate more frequently
than random (Pecinka et al. 2004). It further transpires that
the cohesion between BAC-labelled sister chromatids is
lacking, and that the strict parallel alignment between them
is greater in differentiated and in endopolyploid cells
(Schubert et al. 2006).
Overall, then, CTs in both animals and plants behave as if
they are largely self-organized and independent, influenced
by weak preferences rather than dependent on absolute
configurations. This is perhaps to be expected given the
high levels of copy number variation being found.
2.2.2 Modifications of Chromatin and DNA
One area where plants differ markedly from animal models
of chromatin organization is in the use of the modified
histones which discriminate transcriptionally active regions
16
from those that are silenced, particularly heterochromatin.
Surprisingly the same histone code is not maintained
between animals and plants, or even between plant species
(Fuchs et al. 2006; Shi and Dawe 2006; Braszewska-
Zalewska et al. 2009). Soppe et al. (2002) noted that the
diffuse presence of H3K9 modifications of histone H3 in
euchromatin was characteristic of large genomes, such as
maize, while it was found only concentrated in hetero-
chromatin in Arabidopsis. The ability to redefine such
a fundamental aspect of chromatin organization may reflect
a divergent approach to genome surveillance by plants com-
pared to animals. Plants lack a dedicated germline and the
bulk of silencing signals is maintained at all times, unlike
animals which reset the genome at least twice per generation
(plants do have ‘facultative’ silencing, involved in processes
such as vernalisation and parental imprinting, e.g., Hsieh
et al. 2011). Plant genes are also relatively more compact
and intergenic conserved non-coding sequences (CNS) are
relatively rare. This combination suggests that meristem
integrity may have been protected by enhancing mechanisms
which monitor intergenic regions in order to restrict mobile
element colonisation. In large part this monitoring is carried
out via a small RNA based silencing pathway, involving the
plant-specific RNA polymerases IV and V (RNAP IV, RNAP
V) (Herr et al. 2005; Onodera et al. 2005; Zhang et al. 2007;
Matzke et al. 2009). This represents a significant extension to the
more universal eukaryotic use of different classes of small
RNA to carry out a variety of roles in modulating chromatin
structure and gene expression, for example the use of miRNA
to co-ordinate developmental switches in animals and plants
(reviewed in Kaufmann et al. 2010).
The central role of siRNAs in the control of plant
genome stability has been emerging over the last 10 years
but details about mechanisms are still being revealed.
Briefly, it appears that all eukaryotes are able to detect
‘alien’ transcripts through the recognition of aberrant dou-
ble stranded RNA (dsRNA) structures; the ‘aliens’ are
typically transposable elements (TEs) or viruses but may
include transgenes or even some host genes. Once
recognised, the dsRNA is cut into small fragment(s),
destroying the original transcript but in most cases
retaining a short guide which can be used to recognise
complementary RNA or DNA templates. The ways in
which guides are exploited to modify DNA can vary
greatly; the most extreme use is shown by some fungi, in
which a series of steps leads to mutation of all cognate
sequences (Selker 2002). Plants use a family of Dicer-like
(DCL) enzymes to generate small RNAs from a variety of
dsRNAs which may then be bound by ARGONAUTE
(AGO) proteins and used to direct DNA methylation
(RNA-directed DNA methylation, RdDM) and histone
modification at relevant chromatin sites. Different dsRNA
classes are processed by different, partially redundant,
R.N. Jones and T. Langdon
pathways but the majority of plant small RNAs are 24 nt
long, individually at low abundance and derived from
transcripts generated by RNA polymerases IV and V.
These enzymes supplement the universal RNA polymerase,
Pol II, and produce templates primarily from intergenic and
heterochromatic regions (Zheng et al. 2009) so that the
targets of the 24 nt silencing RNAs (siRNAs) are predomi-
nantly mobile elements, tandem repeats and associated
debris. In addition to transcripts from promoters in the
repetitive elements themselves, there are a range of host
driven transcripts, including for long intergenic non-coding
RNAs (lincRNAs) which may result in almost all of
the genome being transcribed at some point, and hence
potentially able to generate siRNAs. The unusually high
level of retained introns in mature transcripts in plants
(Filichkin et al. 2010) may further add to the pool of
potential silencing signals. The importance of siRNAs is
demonstrated by the presence of a range of additional DCL,
AGO and other associated components, including plant-
specific pathways to generate dsRNA from single-stranded
RNA templates, and to methylate asymmetrical DNA
targets (with the consensus sequence CHH). Plants have
also evolved mechanism(s) to transfer silencing between
cells and tissues (Molnar et al. 2010).
Once symmetrical methylation (at CG and CHG sites) has
been established, it may be maintained at DNA replication
by a hemimethylation recognition mechanism. The 24 nt
siRNAs are required to direct fresh methylation at the asym-
metrical CHH sites, however. This illustrates one of the
features of epigenetic control, that there tend to be multiple
mechanisms which may reinforce each other, but may all be
required for full effect. In combination this may result in
epigenetic haplotypes (‘epialleles’) which are stable for
generations (Johannes et al. 2009; Reinders et al. 2009;
Baubec et al. 2010) but, intriguingly, global responses to
stress may drive some of the initial epigenetic responses,
creating heritable phenotypic variation which may be adap-
tive and allow selection for further epigenetic changes
(reviewed in Boyko and Kovalchuk 2011; Verhoeven et al.
2010; Mirouze and Paszkowski 2011). An interesting model
has suggested that the diversity of DCLs may facilitate the
gradual domestication of particularly adaptive siRNA
sources and their integration into developmental pathway
regulation (Vazquez et al. 2008).
Since methylation and histone modification have longer
range impacts on recombination frequencies and patterns of
DNA replication, and since TE-directed epigenetic marks
may spread to have impacts on adjacent host genes (Gehring
et al. 2009; He and Dooner 2009; Martin et al. 2009), it can
be seen that even in a mature genome, with low levels of
both genic polymorphism and endogenous TE activity,
growing in a relatively stable environment, there is much
scope for divergence of individual lineages. These
2 The Plant Nucleus at War and Peace: Genome Organization in the Interphase Nucleus
constrained conditions rarely apply. Moreover, the predic-
tion that parental conflict over resource allocation in endo-
sperm would result in parent-specific gene expression in that
tissue (Haig and Westoby 1989) is being borne out in
surveys of the distribution of imprinted genes and release
of maternal silencing, while there is also some evidence
for recurring conflicts between divergent centromeric
sequences during meiosis (Malik and Henikoff 2009).
There is therefore intrinsic potential for intragenomic
‘squabbles’ between both conventional and epigenetic
polymorphisms to accumulate over time. In the next section
we consider how frequently evidence for these squabbles
can be seen.
2.3 The Nucleus Under Strain
Some indication of the tolerance required by plant nuclear
organization is provided as soon as a rigorous definition of a
plant species is required. In the extreme this should depend
on strict interfertility criteria but this approach soon runs into
difficulty, both in the inclusion of phenotypically distinct
hybrids, particularly allopolyploids, which suggests the
definition is too broad, and in the exclusion of some clearly
related populations or lines, which suggests the definition is
too narrow (discussed in Soltis and Soltis 2009). Sequence
data do not always resolve these issues (Petersena et al.
2011), and indeed are blurring species boundaries as it
becomes possible to track the history of introgression and
recombination of contrasting haplotypes over million year
periods of time (Wicker et al. 2009). The breadth of intra-
specific phenotypes in the shape of karyotype variants or
crossing incompatibilities has, of course, been known for
many decades. Here we consider some of the genomic
polymorphisms accomodated within a species as generally
understood (Rieseberg et al. 2006), which appears increas-
ingly likely to overlap the variation provided by interspecific
hybridization.
2.3.1 Plasticity of Gene Family Organization
The best defined category of variants could be described as
potential speciation genes (Rieseberg and Blackman 2010)
where polymorphisms at a very limited number of loci are
sufficiently antagonistic that they have a major impact on
fertility. The classic example is hybrid necrosis, where allele-
specific interactions between two loci trigger a disease-like
phenotype leading in the most extreme cases to the death of
progeny. Hybrid necrosis is a problem for most if not all crop
breeding programmes, but is also seen in wild species, occur-
ring in 2% of Arabidopsis intraspecific crosses, for example
(Bomblies et al. 2007). Interactions with a similar
17
maladaptive epistatic allele-specific basis, known as
Dobzhansky-Muller incompatibilities, include hybrid male
sterility in rice (Ouyang et al. 2010), and appear to be rela-
tively more common in plants than animals although whether
this reflects a genuine increase in the number of candidate
genes or an ascertainment bias remains to be established
(Rieseberg and Blackman 2010). At the simplest level,
these interactions may appear to be idiosyncratic, with little
direct relevance to nuclear organization. However, it is useful
to consider hybrid necrosis in some detail as it appears to be
an almost inevitable by-product of the amplification and
diversification of microbial pathogen resistance genes (R-
genes) (Bomblies 2009). Pathogens provide one of the
major selective pressures on plants and models of R-gene
evolution are helping to illustrate the ways in which sessile
organisms can generate the plasticity required within their
nuclear organization to match environmental challenges. It is
likely that this plasticity is required for many other aspects of
adaptation, and will be seen across the nucleus.
There are many R-genes (>150 in Arabidopsis, >400 in
rice) and they are found both singly and in clusters across
genomes. Although they appear to be particularly polymor-
phic (Zeller et al. 2008) and to operate under particularly
strong selection (Chen et al. 2010), the evolutionary dynamics
of their tandemly arranged clusters are similar to those of
other, less well characterised, gene clusters such as those for
F-box proteins (Jain et al. 2007). Tandem duplications of
these genes follow a complex birth-and-death pattern of
evolution, which contrasts with the concerted evolution
of more regular arrays such as rDNA genes (Nei and Rooney
2005). This may be promoted by the recombinogenic effect of
the unpaired additional copy at meiosis (Sun et al. 2008),
enhancing not only ectopic recombination between related
sequences but also using microhomologies to create unusual
chimeras such as the MAF2 alleles affecting Arabidopsis
flowering time (Rosloski et al. 2010). At R-gene clusters
this inherent instability helps to generate the range of defense
options necessary to counter microbial pathogens. Insertion-
deletion polymorphisms (indels) are also common, found at
eleven times the frequency in R-genes as housekeeping genes
in rice and resulting in some 20% of R-genes displaying a
presence or absence variation (PAV) in both rice and
Arabidopsis (Shen et al. 2006). Surprisingly, those loci
showing PAV appear to represent a distinct class which
does not have the high SNP content of other R-gene loci.
Instead they are maintained over relatively much longer
periods, presumably by balancing selection across the popu-
lation (Shen et al. 2006).
Further R-gene complexity is being uncovered in the role
of siRNAs, for example at the Arabidopsis RPP5 region,
which may attenuate expression under normal conditions
but allow release under stress or when pathogens compro-
mise silencing mechanisms (Yi and Richards 2007).
18
An epigenetic component is likely to be a general feature of
R-gene expression, allowing modulation by other biotic and
abiotic stresses (Alcazar et al. 2009), and the development of
immunity (Alvarez et al. 2010) and transgenerational effects on
locus methylation and stability (Boyko et al. 2007). The need
for adaptable regulation is demonstrated by the wide range of
maladaptive phentypes potentially caused by R-gene mutation,
including developmental abnormalities (reviewed in Uchida
and Tasaka 2010) while, more generally, enhanced disease
resistance frequently comes at the cost of other fitness
parameters (e.g., Todesco et al. 2010), leaving the genome
exposed to conflicting pressures. For example, lines exhibiting
hybrid necrosis symptoms at one temperature may be more
stable at another, and may even show greater disease resistance
than sister lines. It is highly likely, therefore, that plant nuclear
organization has had to evolve to permit some chromosomal
regions the ability to create novel gene organization, as has
been suggested to occur for ‘gene nurseries’ in some animal
systems (Nahon 2003; Bailey and Eichler 2006). However,
gene nurseries occur at particular chromosomal contexts and
while there may be analagous regions in plants (Tian et al.
2011), R-gene clusters rarely persist at syntenic locations,
resulting in at least one locus being dubbed ‘nomadic’ (David
et al. 2009). Given these variables, the frequency of maladap-
tive necrotic interactions could be considered rather low!
Another aspect of R-gene evolution that must be
accomodated is the asymmetrical nature of the loci, that is
high levels of PAV and copy number variation (CNV). This
is a pervasive phenomenon in plant genomes (Ding et al.
2007; Springer et al. 2009; DeBolt 2010). The number of
functional genes involved is difficult to quantify accurately
using indirect methods, such as array hybridization, as a
variety of mobile elements have been found to transpose
genic fragments. In maize, fragments of at least 376 different
genes have been duplicated by helitrons (Du et al. 2009)
while in rice fragments of 1,500 genes have been duplicated
by PACK-Mules (Hanada et al. 2009). A very small propor-
tion of these fragments are likely to retain coding function.
Nevertheless, the scale of CNV identified in the most recent
screen of maize (Swanson-Wagner et al. 2010) supports
observations made on smaller sequence-based data sets
(Fu and Dooner 2002; Wang and Dooner 2006) and is
beyond levels expected to be due to genic ‘debris’. Thus,
over 10% of ~32,500 genes surveyed showed CNV, the
majority being absent in one or more lines despite having
Gene Ontology annotations and orthologs in other species.
Most surprisingly, PAV patterns appeared to have an ancient
origin in the maize progenitor, teosinte, and to have
persisted despite domestication and movement into new
environments. It is possible that this represents the presence
R.N. Jones and T. Langdon
of many alternative haplotypes generated as the ancient
palaeoploidy event identified in the maize lineage (Gaut
and Doebley 1997) is resolved by gradual diploidisation,
and less redundant diploid species may not be able to support
such high levels of PAV. Nevertheless, significant levels of
stable CNVs could be generated by relatively mild stress in
Arabidopsis over five generations (DeBolt 2010), affecting
over 1% of total genes. Consistent with the expectation of
tandem gene instability, just over half of genes affected were
in tandemly duplicated regions.
2.3.2 Limits to Expression Neighbourhoods
It is therefore clear that intraspecific crosses must almost
inevitably generate asymmetric CT orthologues. As a conse-
quence it is difficult to envisage mechanisms by which
orthologous loci could be provided with conserved nuclear
environments in which subtle regulatory cues could be con-
sistently supplied. Do individual genes have a high degree of
autonomy in their regulation, to allow them to overcome
shifting levels of neighbourhood-wide expression? Such
autonomy should be encoded within the gene itself (cis-
regulation) but may require more or less strong contributions
from external network-specific factors (trans-regulation).
There has been much recent interest in characterising this
balance and identifying relevant sequences, a field described
as expression or eQTL mapping. A number of studies have
used array hybridization or specific PCR to look at asym-
metrical expression of alleles across the transcriptome in a
variety of plant species. Differences from parental mid-point
values are frequently found, for example at rates ranging
from 4% to 32% of genes in comparisons between
Arabidopsis thaliana ecotypes (Zhang et al. 2008a). How-
ever in Arabidopsis over-dominant expression was seen in
~9% of transcripts examined (Vuylsteke et al. 2005) while a
wider maize study found that the great majority of alleles
were expressed at parental or intermediate levels (Stupar and
Springer 2006). In particular both individual maize parents
frequently showed similar expression levels for a locus; in
all these cases the same level was maintained in the hybrid.
The authors of the maize study concluded that trans-regula-
tion alone was rarely responsible for asymmetric expression,
although cis-regulation frequently was (Stupar and Springer
2006). This fits an alternative view of expressed neighbour-
hoods, in which they are created because of ‘ripple effects’
of read-through transcription outside the target locus
(Ebisuya et al. 2008; Yanai and Hunter 2009). Genes
moved into novel neighbourhoods will adopt the expression
pattern of that neighbourhood to some extent, but cis-acting
2 The Plant Nucleus at War and Peace: Genome Organization in the Interphase Nucleus
elements may allow locus-specific regulation to prevail.
Presumably it is efficient to maintain neighbourhoods of
housekeeping genes, but equally it might be presumed dan-
gerous to fix the expression patterns of loci responsible for,
for example, stress or developmental responses.
2.3.3 Hybrid Vigour
One of the goals of understanding how nuclear organization
ensures optimal plant performance is to improve crop breed-
ing strategies. A particular target is understanding the basis
of hybrid vigour. This occurs when crosses between certain
subsets of a species generate progeny which have far greater
adaptive potential than the additive value of their parents
(Chen 2010). Two contrasting models have been proposed
to account for this. In the dominance model, deleterious
alleles in one parent are complemented by superior alleles
from the other; in the over-dominance model novel
interactions between different parental alleles create
phenotypes not possible from the use of either allele alone.
Despite many decades of use in maize, it has not proved
possible to breed out the putative deleterious alleles of the
dominance model, although there has been a recent claim
that vigour has been dissected into a small number of
complementing QTL (Sch€ on et al. 2010). Now, a recent
maize resequencing project has provided evidence that
absent PAV loci are being complemented by PAV present
loci from the other parent (Lai et al. 2010 - note that the
resequencing approach provides a more conservative esti-
mate of ~300 genes present in the B73 reference but missing
in other lines). This is consistent with the lack of significant
over-dominant expression patterns in hybrids, although
some component of vigour could still be contributed by
such interactions (Stupar et al. 2008).
2.3.4 Mobile Elements
Brief mention should also be made of the very large contribu-
tion that mobile or transposable elements (TEs) make to
intraspecific variability. Variation in mobile element profiles
across, and even within, populations has been well
documented and has frequently provided the basis for
characterising crop genetics and domestication histories
(Kalendar et al. 2011). As an example of the extent of TE
content variation, two sequenced maize genomes differ in size
by ~20%, mostly ascribed to differences in TE content
(Vielle-Calzada et al. 2009). As an example of TE links to
host phenotype, variation in the BARE-1 retrotransposon
correlated with adaptation to local environmental conditions
in Hordeum spontaneum (Kalendar et al. 2000). Across a
300 m transect, BARE-1 copy number varied three-fold.
19
Perhaps most surprisingly, plants in the most arid and inhos-
pitable conditions exhibited both the highest numbers of
BARE-1 elements and the greatest retention of BARE-1
sequences, despite the naive expectation that these properties
would reduce fitness. TE profiles do not, therefore, automati-
cally conform to intuitive expectations although a general
correlation of expression with stress responses is found,
coupled with a propensity to act as nucleation points for
epigenetic effects which spread into adjacent host genes.
Ecological analogies for TE behaviour appear to be apt
and descriptions of the opportunistic nature of niche exploita-
tion by specific element types or sub-families abound
(see Kejnovsky et al. 2012; Slotkin et al. 2012). A crucial
point, however, is that horizontal transfer of TEs is not suffi-
ciently widespread in plants to provide a reliable escape
mechanism such as may be argued to occur, for example, in
Drosophila (Bartolome et al. 2009); the fates of plant TEs are
therefore tied to those of their hosts and in the long-term their
behaviour cannot afford to be as destructive as, say, viral
retroelements. As part of the restraining process acting
on TE ‘selfishness’, non-autonomous elements frequently
mediate the most significant phenotypic effects of TE families
on their hosts but also attenuate the activity of rare active
autonomous elements and help to disperse their impact across
populations. The variability created by TE activity is not
restricted to direct impact at insertion sites. A good recent
example of their general ability to remodel expression
demonstrated that TE proximity reduced Arabidopsis gene
expression (Hollister et al. 2011); the strength of this effect
depended on both the host species and the frequency with
which the TE was targeted by siRNAs.
2.4 The Nucleus at War
The previous section outlined the sometimes surprising levels
of structural variation that may be found between individuals
of the same plant species. Not all variation within a species
may be accommodated in any single cross, and not all intra-
specific incompatibilities may be ascribed to such relatively
simple causes as hybrid necrosis (e.g., Zhao et al. 2010).
However, by definition, it might be expected that the great
majority of genomic tensions would be held in check within a
species. As McClintock (1984) pointed out ‘species crosses
are a potent source of genomic modification. .. the alterations
produced when the genomes of two species are combined
reflect their basic incompatibilities’. New molecular tools
are now providing us with the means to investigate what
happens when the genomes of different species, as F1 hybrids
or allopolyploids, come together for the first time within
a single nucleus (Hegarty and Hiscock 2005) and in the
common cytoplasm from the maternal parent (Fig. 2.2).
What happens, then, once there is no longer a need for
20
Fig. 2.2 Diagrammatic representation of the plethora of interactions which can occur when genomes of different species are combined together
within a single nucleus, and within the cytoplasm of the maternal parent (partly based on Gill 1991)
genomes to be compatible in shared germplasm? Do interspe-
cific hybrids require extreme chance or strong selection for
divergent genomes to cohabit, or will the forces constraining
normal sibling jostling continue to maintain common
standards of organization and expression in the absence of
routine requirements for interfertility? In this section we
review the adjustments found to occur in interspecific
hybrids, both where genomes are sufficiently similar to
allow homoploid formation, and the far better characterised
situation where each parental genome persists separately as a
component of a new polyploid. Sometimes hybridization
appears to proceed relatively smoothly, while in other cases
one genome appears to fall victim to another. We are far from
understanding the processes responsible for these differences,
although some common patterns appear to be emerging.
2.4.1 Ploidy Levels
Homoploid hybridization generates novel mosaic genomes, in
which some proportion of each parent must be lost. The gene
content and expression patterns of each parental genome
should therefore be relatively well conserved. It might also
be expected that parental chromosomes must show extensive
colinearity to allow reciprocal recombination. Consequently
there is likely to be a limit to the amount of divergence that can
be successfully tolerated for the homoploid generation. The
parameters of this limit have been vigorously discussed
R.N. Jones and T. Langdon
(Buggs et al. 2009; Paun et al. 2009). Very detailed work
has been carried out on a number of young homoploid
hybrids (Baack and Rieseberg 2007), but higher sequencing
throughput is likely to throw up multiple examples of
paleohomoploidy, with signs of suspect ancestry already
showing in model systems, including an Arabidopsis relative
(Wang et al. 2010) and Brachypodium distachyon (Wolny
et al. 2011).
Polyploidy requires an additional genome doubling step
compared to homoploid hybridization but allows cohabita-
tion of far more divergent genomes. It not only retains all
parental material at initial stages, so that parent-specific
processes may potentially be maintained, but it also provides
redundancy such that maladaptive interactions may subse-
quently be overcome by silencing or deletion rather than
requiring specific adaptation. From the previous sections it
can be seen that plant nuclear organization is already primed
to accomodate ‘alien’ CTs and unstable gene neighbour-
hoods, and to identify and suppress loci producing aberrant
transcripts, so it is not surprising that successful polyploi-
disation events occur frequently. There is evidence for them
having occurred in more than 70% of species at some stage
during their evolution (Levin 2002; Fawcett et al. 2009), and
it has been suggested that polyploidy may even be ubiqui-
tous among angiosperms (Soltis et al. 2009; Jiao et al. 2011).
Autopolyploids have multiple sets of chromosomes from
a single species, and are of interest because they allow
examination of the simple effect of increasing CT numbers
2 The Plant Nucleus at War and Peace: Genome Organization in the Interphase Nucleus
in the nucleus. As might be expected, only relatively subtle
effects on expression are usually seen (Stupar et al. 2007;
Parisod et al. 2010) although in the longer term differentia-
tion of the chromosomes occurs, mostly by loss of material
and presumably to reduce irregularities at meiosis. The
majority of polyploids are formed between divergent species
(allopolyploids) and conflicts might therefore be expected at
hybridization due to differences in genome size, genome
composition, regulatory mechanisms, cell cycle duration,
genetic and epigenetic modifications and indeed all of the
aspects that contribute to harmony in the physiology of the
diploid or autopolyploid nucleus. A large number of potential
conflicts must be resolved rapidly, typically within a genera-
tion or two, a scale which dictates extensive use of epigenetic
regulation. This phase of polyploidisation has been called
‘revolutionary’, in contrast with the much longer ‘evolution-
ary’ phase during which the new species progresses back
towards diploidy (Levy and Feldman 2002); this phase
appears to still be ongoing in maize at least five million
years since a tetraploidy event (Schnable et al. 2011).
2.4.2 Changes at the Gene Level
Newly synthesised allopolyploids and F1 hybrids are subject
to new combinations of regulatory networks, as well as rapid
genetic modifications which can result in altered patterns or
levels of transcription. The extent of these interactions will
depend on the degree of divergence between the progenitors.
Recent advances in molecular techniques have now enabled
researchers to directly compare new hybrids and their
parents; and the impact of hybridization and genome dupli-
cation at the genic level can involve sequence loss, changes
in transposable elements, altered patterns of gene expression
and epigenetic modifications. These changes have been dealt
with in detail in Jones and Hegarty (2009), and are consid-
ered here only briefly.
2.4.2.1 Sequence Loss
Over time polyploids are known to lose redundant
parental sequences, a process known as fractionation or
diploidisation. However, sequence loss can occur surpris-
ingly rapidly, as was first reported for newly formed
allopolyploids in Brassica (Song et al. 1995), followed
soon after by Triticum aestivum (Feldman et al. 1997).
These early reports used restriction analysis and Southern
blotting to detect changes in low-copy sequences. There
were some differences between the species, with Brassica
instabilities continuing beyond the fifth generation while
wheat stabilised by the second or third allopolyploid gener-
ation (Ozkan et al. 2001; Shaked et al. 2001). Strikingly,
there was evidence both for preferential loss of material
21
from specific genomes, and for recapitulation of at least
some of the changes found to have occurred in natural
polyploids (Shaked et al. 2001). Genome-wide characterisation
of both newly synthesised and ancient allopolyploids is now
possible at the sequence level and it is apparent that preferential
loss is a general phenomenon. In maize, there is evidence that
one parental genome was not only the preferential target for
initial sequence loss, but that this preference has persisted over
millions of years of subsequent evolution (Schnable et al.
2011). There is also evidence for preferential loss of particular
TE sub-families (Kraitshtein et al. 2010; see below). The
mechanism(s) responsible for this loss are unknown but a
recent review of Triticeae polyploids found evidence that the
largest genome in the hybrid was most likely to suffer loss
(Bento et al. 2011). However, experimental conditions or
intraspecific variation among parents may have a strong
influence on this behaviour, as Mestiri et al. (2010) found no
evidence for any rearrangement in a number of synthetic wheat
allohexaploids.
2.4.2.2 Changes in Transposable Elements (TE)
Widespread changes to the activity of TEs were proposed by
McClintock (1984) as a likely consequence of the ‘genome
shock’ induced by the merger of two divergent genomes
in a single nucleus; and experimental studies have now
confirmed that proliferation of TEs occurs in newly formed
diploid hybrids of Helianthus (Ungerer et al. 2007) and
contributes to genome size expansion in these taxa. The
activation of normally silenced retrotransposons has also
been demonstrated in synthetic allotetraploid wheat
(Kashkush et al. 2002), but in this case no accompanying
burst of transposition was observed (Kashkush et al. 2003).
Surprisingly, analysis of the history of retrotransposon
colonisation of the wheat genomes also found that prolifera-
tion rates were neither enhanced nor repressed by polyploi-
disation (Charles et al. 2008). A more recent paper from the
Kashkush group (Kraitshtein et al. 2010) examined the
behaviour of a specific TE sub-family, the Veju TRIM
retrotransposons, in a new allohexaploid. Veju was chosen
as a high copy functional family, expected to be particularly
sensitive to epigenetic changes. Over 50% of Veju sites
showed altered methylation, in most cases a reduction, in
early generations, returning to normal or hypermethylated
levels by S4. Unexpectedly there was widespread loss of the
elements in the first generation, partly compensated by a
subsequent increase in copy number. As with sequence loss
(above), TE modification may be strongly dependent on the
genetic backgrounds or ploidies of the hybrids used; the
Kashkush group found hypermethylation of class II TEs
(DNA transposons) in synthetic allohexaploids, but
hypomethylation in synthetic allotetraploids (reviewed in
Yaakov and Kashkush 2011).
22
2.4.2.3 Altered Gene Expression in Allopolyploids
Changes in gene expression appear to occur during both
revolutionary and evolutionary phases of polyploidy. While
the immediate (revolutionary) selective pressures might be to
minimise deleterious expression, in the long term the pres-
ence of duplicate loci may permit the evolution of homoeologs
with different functions. Some of the most detailed
transcriptome work has been carried out in Gossypium,
which includes allotetraploids combining Old and New
World lineages in hybridization events some one to two mil-
lion years ago. Gene deletion is very rare but there appears to
be a constant bias for the polyploids to recover the transcrip-
tion pattern of the New World lineage (D genome) whether
this be for higher or lower levels of expression than seen in the
Old World lineage (A genome) (Flagel and Wendel 2010).
Synthetic Gossypium allopolyploids suggest that bias is par-
ticularly high during the ‘revolutionary’ phase (Rapp et al.
2009) and that it occurs even in an F1 hybrid where no change
in ploidy is involved (Flagel and Wendel 2010). The ‘evolu-
tionary’ phase of the natural allopolyploids therefore appears
to have rather equalised the contribution of each parent, par-
tially reversing the initial bias (Flagel and Wendel 2010). As
predicted, there is good evidence that at least some
homoeologous loci have evolved differential expression
patterns (Dong and Adams 2011).
Other key work on altered patterns of gene expression
in allopolyploids has come from studies on Arabidopsis.
Synthetic lines of allotetraploid Arabidopsis suecica, using
oligonucleotide arrays, showed non-additive changes to the
expression of approximately 5% of genes represented on the
array (Wang et al. 2006); and around 56% of these genes
were affected in a similar way in two independently
generated lines. Wang et al. (2006) showed that the affected
genes did not appear to be limited to any particular chromo-
somal region, and belonged to a broad range of functional
categories. An excellent recent paper (Chang et al. 2010)
may have resolved these complexities and provides a model
against which to gauge other systems. A tiling array with
over 400k features, confirmed by resequencing, was used
exhaustively to uncover a pattern in which interacting
networks of genes derived from a single parent appeared to
have been selected. Heterologous networks were signifi-
cantly under-represented and the loci from the Arabidopsis
thaliana lineage also tended to be expressed less frequently
than those of the A. arenosa lineage. The authors propose a
number of explanations for these findings, which are not
mutually exclusive. Firstly, the A. thaliana genome may be
disadvantaged both by its selfing history and by the fact that
its native range is to the south of both A. arenosa and the
hybrid, exacerbated by the possibly fortuitous predominance
of A. arenosa-derived components in the hybrid’s transcrip-
tion machinery. Second, selection for networks derived
R.N. Jones and T. Langdon
from a single parent would minimise the expression of
Dobzhansky-Muller incompatibilities, such as hybrid necro-
sis as discussed above. Such selection could also follow
from disruption of expression neighbourhoods containing
genes in the same network. Observations from a number of
systems suggest that fractionation tends to be clustered,
which might simply represent elimination of neighbour-
hoods damaged by a random initial event.
Similar analysis could be applied to data reported in other
allopolyploid systems such as Senecio (Hegarty et al. 2006;
Hegarty et al. 2008), and wheat (Pumphrey et al. 2009). This
topic is explored in much greater depth in Jones and Hegarty
(2009).
2.4.2.4 Epigenetic Changes
Much of the initial research into gene silencing in
allopolyploids was performed using synthetic lines of
Arabidopsis suecica, which displayed considerable pheno-
typic variation in the F2 generation and several cases of
unstable phenotypes compared with the F1 (Comai et al.
2000). cDNA-AFLP analysis showed that a number of
genes were potentially silenced in the allotetraploid
lineages; and Southern blot assays using methylation-
sensitive restriction enzymes indicated that these genes
were methylated in the hybrids. It appeared, however, that
there were no gross changes to the overall level of cytosine
methylation in the allotetraploid genome (Madlung et al.
2002); but the use of methylation-sensitive AFLP (MSAP)
showed that 8.3% of loci scored showed differential
methylation between the parents and the F3 synthetic
allotetraploids. The majority of these (76.9%) were consis-
tent across the four allotetraploid individuals studied, with
62.5% of this group showing demethylation and the remain-
der displaying an increase in methylation (Madlung et al.
2002). These results suggest that the methylation pattern of
allopolyploids is subject to widespread modification in a
concerted manner. The role of small RNAs in allopolyploid
systems is only now coming to light, although theories
suggest that incompatibilities between divergent parental
genomes could result in three specific effects in polyploids.
The interaction of the two (or more) genomes could result in
either (1) changes to the accumulation of siRNAs and
miRNAs, or (2) changes in the efficiency of the siRNA/
miRNA biosynthetic machinery, or (3) alterations to the
specificity of the targets (Chen and Ni 2006). In the first
such scenario, transcription will be affected differently
depending upon the fidelity of the small RNAs for their
targets. Low fidelity, where small RNA accumulation is
increased (due to ploidy change), will result in silencing/
downregulation of both parental transcripts, but high fidelity
will affect only targets from one parent. This topic is also
covered in greater detail in Jones and Hegarty (2009).
2 The Plant Nucleus at War and Peace: Genome Organization in the Interphase Nucleus
2.4.2.5 Non-destabilised Genomes
Finally it should be noted that not all detailed studies find
major genome shocks to occur on hybridization (e.g.,
Ammiraju et al. 2010). A screen of ~22,0,000 loci in cotton
allopolyploids failed to find evidence for sequence or meth-
ylation changes, nor were gross changes in TE content
detected (Liu et al. 2001), suggesting that the expression
changes described above could have been mediated by ‘nor-
mal’ pathway interactions. In support of this, a recent com-
parison of domesticated and wild Gossypium species
suggests that domestication alone has led to alterations in
expression of almost a quarter of transcripts in fibre cells,
without recourse to ‘genomic shock’ (Rapp et al. 2010).
2.4.3 Adjustments at the Chromosome Level
Insights into genome organization and evolution at the
sequence level are now based on comprehensive data sets,
with a flood of comparative information anticipated over the
next few years. Polyploids remain difficult for current
technologies but clear pictures of the extent of sequence
change induced by hybridization, as opposed to normal
wear and tear, can be expected. Possibly the greatest chal-
lenge for the future, however, will be to understand how
nuclear organization at the chromosome level is maintained
and adapts in the face of sequence change, particularly the
large structural variations and dynamic shifts in gene content
and activity described above. These descriptions did not
extend to other aspects of chromosome organization which
are also subject to selection, such as variable recombination
rates, maintenance of synteny, replication origins and so
forth. As discussed for CTs, a strong self-organizing poten-
tial must be involved, as well as a high degree of plasticity.
These properties seem to hold within a species, and intraspe-
cific chromosomal polymorphisms are usually rather
limited, in contrast to the sequence-level ‘strains’ outlined
in Sect. 2.3. Hybridisation can lead to wholescale and often
rapid reorganization, however. In the final section we will
discuss how chromosomes adapt (or not) when they fall out
with the new neighbours in the nucleus.
The process of adjustment leading to the establishment
of stable polyploids can involve random translocations
as well as species-specific rearrangements in particular
chromosomes in all polyploid populations of a species
(Leitch and Bennett 1997; Lim et al. 2008; Wright et al.
2009). Studies in newly formed or experimentally induced
allopolyploids give us insights into the scope and the timing
of these events, be they stochastic or otherwise. The pairing
of chromosomes at meiosis can be compromised, especially
in F1 hybrids, sudden changes in histone codes can deter-
mine how DNA is organized and how sequences are
expressed in chromatin, and the way in which DNA is
23
embedded in chromatin, and these conflicts in turn must
impact on the way in which genomes interact and accom-
modate to sharing a nucleus (Jones and Pašakinskienė 2005).
There may also be problems for the spatial separation of
genomes and the arrangements of nuclear territories.
Centromeres may be under separate genetic control, and
those from one genome can suffer suppression and silencing
by the other. This may lead to chromosome instabilities,
even at a tissue-specific level, with the whole genome or
part of the genome being eliminated as the response. The
balance of chromatin may also change, with one species
coming to sequentially represent a larger component of the
genome than its partner over successive generations.
Intragenomic recombination may also remodel a hybrid, as
explained below.
2.4.3.1 Chromosome Changes in F1 Hybrids
F1 hybrids have serious problems in dealing with chromo-
some pairing. There may be differences in chromosome num-
ber as well as chromosome size, and even where there is some
balance between the species involved there is frequent forma-
tion of univalents and lack of viable gametes. Nothwith-
standing these conflicts there are some striking examples of
their success in dealing with their hybridity. Asexual re-
production, as in the Egyptian tree onion, 2n ¼ 2x ¼ 16,
Allium cepa  A. fistulosum (Hanelt 1990; Jones 1991), is a
prime example. Bulbils form in place of flowers. Apomixis is
also a common means by which F1 hybrids can persist
(Rieseberg 1997).
A large difference in genome size, based on the measure-
ment of nuclear DNA amounts, as in the FI hybrid, is not
necessarily a factor in creating genome conflict. The hybrid
ryegrass, Lolium temulentum  L. perenne (2n ¼ 2x ¼ 14),
for example, has two sets of chromosomes which are struc-
turally and genetically dissimilar, differing in DNA amounts
by about 50%, and yet they form a stable F1 hybrid with
regular bivalents at meiosis and with chiasma frequencies
which are similar to those of the parents, although the fertility
is low. This hybrid has the capacity to resolve differences
in terms of its synaptonemal complexes and to produce
homoeologous bivalents with functional and morphological
integrity (Jenkins and White 1990). This raises the question –
if genome or chromosome size is not a critical factor then
what is it that makes for conflict? The answer it seems is that
structural differences between chromosomes are not neces-
sarily size-dependent. Hybrid systems are known where
translocations or tranversions may be the main cause of loss
of fertility in an F1 hybrid (Rieseberg 1997).
2.4.3.2 Chromosome Elimination in F1 Hybrids
The extreme of the conflict in F1 hybrids is where one
parental genome is rapidly eliminated by the other due to
mitotic processes. This often happens in the first few nuclear
24
divisions of the zygote; although in some cases where
perenniality is involved the elimination may be incomplete.
The phenomenon was first correctly interpreted in Hordeum
vulgare  H. bulbosum by Kasha and Kao (1970), and is
now known in many other interspecies and intergeneric
crosses.
The essential components of the system can be briefly
summarised as follows:
(1) Species vary in the extent of the elimination involved.
In some hybrids stable F1 plants may be established,
while in other genotypes there is elimination of one
of the parental genomes, and when it does occur the
extent varies, so that some plants can remain as mosaics
for long periods of time, e.g., H. vulgare  H. bulbosum
(Thomas and Pickering 1983; Riera-Lizarazu et al.
1996).
(2) Eliminated chromosomes often fail to congress on to the
metaphase plate or to reach the anaphase poles during
the early divisions of the zygote when the elimination is
taking place, as in H. vulgare  H. bulbosum (Bennett
et al. 1976). Failure to congress could be due to a lack of
efficiency in attachment to spindle microtubules; and as
later studies in wheat  maize crosses demonstrated, all
of the much smaller maize genome chromosomes were
lost during the first three cell divisions (Laurie and
Bennett 1989), or early embryogenesis (Mochida et al.
2004) in most of the embryos. The maize centromeres
were either tiny or non-visible and without affinity for
spindle attachment, and the maize nucleolar organizing
regions (NORs) were also suppressed (Laurie and
Bennett 1989). The same is true in wheat  sorghum
crosses (Laurie and Bennett 1988). In barley  maize
hybrids the maize chromosomes in the zygote had well
defined centromeres, but even so they were still
eliminated (Laurie and Bennett 1988).
(3) In stable hybrids the genomes may show some level
of spatial separation, as in H. vulgare cv. Tuleen
346  H. bulbosum (Anamthawat-Jonsson et al. 1993),
and the differential behaviour of the parents indicated
that their centromere activity must be under separate
genetic control. In later studies on the same hybrid
Schwarzacher et al. (1992) confirmed genome separa-
tion, as well as the near identical size of the
two genomes. Centromere-associated structures of
H. vulgare were larger than those of the more peripheral
H. bulbosum chromosomes which have ‘weaker’
(smaller) centromeres. Genome separation has also
been found in several other hybrids (Finch et al. 1981;
Leitch et al. 1990; Leitch et al. 1991).
(4) In the hybrid H. vulgare cv. Tuleen 346  H. marinum
elimination of the Tuleen 346 genome occurs in the endo-
sperm, while that of H. marinum is lost in the embryo; i.e.,
R.N. Jones and T. Langdon
there is tissue-specific ‘alternative elimination’ of parental
genomes (Finch 1983; Finch and Bennett 1983). The
eliminated chromosomes again had smaller centromeres,
and tended to occupy more peripheral positions. The
authors suggested tissue-specific suppression of genes
for centromere function. Interestingly, the eliminated
chromosomes also showed suppression of their NORs.
(5) The most compelling evidence we have to date that the
centromere underlies chromosome instabilities in
hybrids comes from the application of DNA fibre-FISH
to addition lines of individual maize chromosomes
added to oat (Jin et al. 2004). The centromeric DNA
(CEN-DNA) is intermingled with centromere-specific
retrotransposons (CRM), and these two components
make up a range of sizes varying between ~ 300 and
2,800 kb for individual maize centromeres. The point of
interest is that in addition lines with two different genes
coding for the CENH3 histone, it is the oat gene that is
dominant and the oat CENH3 that becomes incorporated
into the maize centromeres. The maize CENH3 gene is
silenced, and the oat CENH3 organizes the kinetochore
on the maize chromosomes. Interestingly, in oat 
maize hybrids it is the maize chromosomes which are
eliminated and the maize centromeres which are
impaired in the hybrids. In the oat  maize crosses the
elimination of the maize chromosomes is more gradual
than it is in crosses such as wheat  maize and barley 
maize (Riera-Lizarazu et al. 1996), but nonetheless it
does occur, and the least we can now say is that the
centromeres find themselves compromised in hybrids
and are subject to suppression and silencing together
with the NOR ribosomal RNA genes.
In wheat  pearl millet hybrids Gernand et al. (2005) have
shown how the pearl millet chromosomes in young embryos
occupy a distinct interphase territory, mainly at the periphery of
the nucleus. The pearl millet chromatin is then sequentially
eliminated, over successive mitoses, by the formation of
micronuclei which bud-off from the wheat nucleus. At the
same time there is failure of segregation of pearl millet
chromosomes at mitosis itself as well as obvious changes to
their structural integrity. Thus there are two processes going on
which gradually eliminate all the pearl millet chromatin, which
is then degraded and lost, leaving the haploid wheat nucleus.
The authors further speculate that the elimination process
involves differences between wheat and pearl millet in post-
translational histone modifcations; but such differences were
not found in Hordeum marinum  H. bulbosum hybrids and
their parents (Sanei et al. 2010).
New data from Ishii et al. (2010), using in situ
hybridization to probe both genomic DNA and centromere-
specific repeats, confirm that pearl millet chromosomes are
eliminated in crosses with several members of the Triticeae.
2 The Plant Nucleus at War and Peace: Genome Organization in the Interphase Nucleus
The elimination process showed several of the features
previously described by Gernand et al. (2005), including
chromosome breakage, nondisjunction, micronuclei forma-
tion and failure of chromosome segregation at mitosis. Ishii
et al. (2010) further revealed that the cause of elimination
cannot be explained by malfunction of kinetochores binding
to spindles, but proposed that it could be due to differences
in the properties of cohesins between sister chromatids in the
wheat and pearl millet chromosomes. In hybrids with oat
however, all seven of the pearl millet chromosomes were
retained, except for a small percentage of cells with
decreased (6.1%) or increased (2.4%) numbers of pearl
millet chromosomes, and carried centromere signals of the
pearl millet chromosomes.
2.4.3.3 Chromosome Changes in Allopolyploids
New hybrids arising by allopolyploidisation, either through
genome duplication of an existing F1 or through the union of
unreduced gametes, are immediately subject to a strong form
of reproductive isolation, and allopolyploidy is therefore a
direct route to instant speciation (reviewed in Hegarty and
Hiscock 2008). The allopolyploid ‘marriage’ however is not
harmonious: it results in an irreversible burst of reorganiza-
tion and modification of the genomes involved. There are
immediate and genome-wide changes at the visible level of
chromosomes and phenotype, as well as cryptic
modifications involving gene expression mediated by
genetic and epigenetic mechanisms caused by alterations in
gene regulatory networks (Matzke et al. 1999; Riddle and
Birchler 2003).
Changes at the whole genome/chromosome level can be
visualized using genomic in situ hybridization (GISH)
(Pašakinskienė and Jones 2005); and this differential ‘paint-
ing’ of chromosome sets is a useful cytological resource for
studying genome conflict in hybrids and allopolyploids. An
interesting and novel case involves a hybrid between Lolium
multiflorum and the allopolyploid Festuca arundinacea:
itself a natural allohexaploid originating as a hybrid between
F. pratensis and F. glaucescens (genome composition
FpFpFgFgFgFg, Humphreys et al. 1995). In colchicine-
doubled F1 C0 octoploids of L. multiflorum  F. arundinacea
(2n ¼ 8x ¼ 56) some genotypes, as revealed by GISH,
restructured themselves as ‘novel diploids’ by diploidisation
(i.e., becoming diploid, 2n ¼ 14) and somatic recombina-
tion (Pašakinskienė et al. 1997). Using GISH these new
genomic variants were shown to contain components
derived from F. pratensis, L. multiflorum and F.
glaucescens, with genomes of the three species being present
in different proportions and as variable patterns, and with F.
pratensis chromatin as their genomic basis.
Similar events were recently found in a selected F1 C1
hybrid from the same population of plants. A hexaploid
genotype F2 3–18 (2n ¼ 6x ¼ 42) was characterised as a
25
‘super-recombinant’, with a high number of recombinant
chromosomes. Some of these new chromosomes were
constructs composed of chromatin from the three component
species L. multiflorum – F. pratensis – F. glaucescens. This
genotype was observed for a number of years, and was found
to have high vigour complemented by a good level of fertility.
Its instability became apparent in phenotypic segregation,
during vegetative multiplication. In a few cases the initial
hexaploid gave rise to somatic segregants with a diploid
chromosome number of 2n ¼ 2x ¼ 14 (Pašakinskienė and
Jones 2005).
The instabilities described above in the L. multiflorum 
F. arundinacea hybrids are genotype-specific, and the plants
which became diploid (2n ¼ 2x ¼ 14) are constructed de
novo from the tri-specific genomic conflict involving
L. multiflorum – F. pratensis – F. glaucescens. In most
cases the chromosomes of the novel diploid are very similar
to the chromosomes of pure F. pratensis diploid, but in some
cases the F. pratensis genome has gained various-sized blocks
of L. mulitflorum chromatin, and sometimes F. glaucescens is
present but not in all of the segregants. In any case the
reconstructed chromosomes are different from those existing
in the F. pratensis genome within F. arundinacea, according
to their GISH-banding pattern (Pašakinskienė et al. 1998).
It is assumed that the ‘novel diploids’ could have resulted
from concerted translocations, where at some stage the
entire newly made allopolyploid genome was a ferment of
rearrangements of its constituent species-specific parts. The
centromeres have most likely also played an important role in
the formation of these ‘novel diploids’. We speculate that the
centromeres could be ‘novel’ as well, built on the basis of the
centromeric components of the parental species involved in
the chromosome set of the hybrid genome. A similar story to
Lolium – Festuca, but involving meiotic as well as mitotic
recombination, has recently been reported for a derivative
reconstructed ‘Zebra’ chromosome from an Elymus
trachycaulus  Triticum aestivum hybrid (Zhang et al. 2008b).
2.4.3.4 Genome Drift in Festulolium
Another manifestation of ‘genomes at war’ is revealed by
changes to ‘genome balance’, or ‘genome drift’ in otherwise
stable Lolium-Festuca allopolyploids. In the F8 population
of the tetraploid hybrid ‘Prior’, L. perenne  F. pratensis,
meiosis was observed to be stable in the early generations,
with a high level of bivalent formation. However, a GISH
study revealed that extensive recombination had taken place
between homoeologs of the two genomes, and that the bal-
ance of chromatin was not equal (Canter et al. 1999). The
substitution of Festuca-origin chromosomes by those of
Lolium-origin resulted in a mean of 17.9 Lolium and 9.7
Festuca chromosomes per genotype. These results are simi-
lar to those of an earlier study looking at another hybrid
L. multiflorum  F. pratensis (Zwierzykowski et al. 1998).
26
Fig. 2.3 Changes in the proportion of parental chromatin in the
allotetraploid Festuca pratensis  Lolium perenne between generation
F2 (left) and F6 (right) using genomic in situ hybridization. L. perenne
chromosomes are labelled yellow whereas those originating from
F. pratensis appear orange. Recombinant chromosomes are indicated
by arrows, and the genomes are virtually identical in size
Here it was shown that the proportion of the genome
occupied by L. multiflorum chromatin ranged from 49.2%
to 66.7%, and this likewise confirms the balance in favour of
Lolium over Festuca. Kopecký et al. (2006) have now shown
that the phenomenon is widespread within the Festulolium
species complex. They used GISH to study the genomic
constitution of more than 600 plants from virtually all com-
mercially available Festulolium cultivars, and found a large
range of variation in the proportions of parental genomes
and the level of intergenomic recombination, including
several cultivars which were at the F9–F10 generation.
The reasons for the dominance of Lolium over Festuca in
this way are not understood. Various theories have been
proposed, such as gametic competition, pollination effects
or selection for vigour in the early stages of seedling growth,
but no definitive answers have yet emerged. In the light of
recent knowledge of the centromere organization and func-
tion we could conjecture that the Lolium centromeres are
more competitive than those of Festuca, and this may
account for the predominance of Lolium chromatin in these
hybrids, although any convincing mechanism still awaits
discovery.
A similar story to that of Festulolium was earlier described
by Anamthawat-Jonsson (1999). Using GISH she recovered a
unique set of chromosomes in Triticum (2n ¼ 4x ¼ 28,
AABB)  Leymus (2n ¼ 4x ¼ 28, NNXX) hybrids. The
allopolyploids stabilised over a number of years as a set of
six pairs of Leymus and 15 pairs of wheat chromosomes
(Anamthawat-Jonsson 1999). The unique composition proba-
bly resulted from the stable replacement of one pair of Leymus
chromosomes by the addition of one pair from wheat, together
with the selective elimination of eight pairs from Leymus.
It is clear the conflicts and chromosome instabilities in
allopolyploids have a higher degree of complexity and a larger
variety of outcomes. There are more conflicts to resolve and
more ways in which resolutions can occur.
Recent studies involving allotetraploids of Festuca
pratensis  Lolium perenne have now tracked changes in
the balance of chromatin in somatic cells over six successive
R.N. Jones and T. Langdon
generations of open pollination (Zwierzykowski et al. 2006),
and the outcome is the same (Fig. 2.3.). The proportion of
total genome length contributed by L. perenne chromatin
increased from about 50% in the F2 to about 60% in the F6,
raising speculation about how far this chromatin replace-
ment could go over further cycles of open pollination. The
mechanism therefore appears to be progressive by small
incremental shifts at each generation.
A key question in all of these studies revolves around the
use of GISH, the level of resolution and how to interpret the
observations. The sensitivity of GISH is limited to large
megabase-sized chromosome segments (Lukaszewski et al.
2005), which could explain the imbalance in genome
proportions of the hybrids, but this still leaves open the
mechanism by which GISH megabases change. A new
approach to dealing with this question has been to up the
level of resolution of genome structure using Diversity
Array Technology with polymorphic DArT markers, which
allows for the screening of several thousands of markers at a
single pass (Kopecký et al. 2009; Kopecký et al. 2011).
Species-specific markers were identified which yielded
results at variance with the GISH work, but the study also
confirmed the enhanced sensitivity of the method and its
potential for future analysis of ‘genome drift’.
2.5 Concluding Remarks
The story of order and chaos in the plant nucleus is incom-
plete and open-ended. The knowledge that we have is based
on only a handful of species, including model organisms
with small genomes and a few plants of economic signifi-
cance such as maize, rice and wheat. Nonetheless, certain
observations have emerged which are both surprising and
confusing. There is no normal or fixed order for the structure
of the interphase plant nucleus; it depends on genome size
which has a seamless array of values with no cut-off point
between what is a small and large genome, and what
happens to the order of the nucleus across this continuum
of sizes. Nature has dealt with the advent of allopolyploidy,
and experimental studies on nascent allopolyploids confirm
the strong barriers that have had to be rapidly overcome in
the process. There is a rich harvest of new discoveries yet to
be made on order and chaos in the plant nucleus, and in the
process of doing so we still need to bear in mind recent
findings (e.g., maize and Arabidopsis) that many diploids,
if not all of them, have a history of having passed through
earlier cycles of ploidy events, and still bear the duplications
as evidence. As far as hybrids are concerned, our new
discoveries on genome readjustment have implications for
the fundamental understanding of genome change in evolu-
tion, as well as presenting opportunities for the release of
new forms of genetic and epigenetic variation in crop plants.
2 The Plant Nucleus at War and Peace: Genome Organization in the Interphase Nucleus
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 The Organization of Genomic DNA in Mitotic
Chromosomes: A Novel View 3
Hideaki Takata, Sachihiro Matsunaga, and Kazuhiro Maeshima

Contents 3.1 Introduction

3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2 Chromatin Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
In 1878, W. Flemming discovered a nuclear substance that
3.2.1 Nucleosome Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 was visible on staining under the light microscope and named
3.2.2 30-nm Chromatin Fibre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 it ‘chromatin’, which is the basic unit of genomic DNA
3.3 Structure of Mitotic Chromosomes . . . . . . . . . . . . . . . . . . . . . . 35 organization (Olins and Olins 2003). During cell division,
3.3.1 Classical Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 chromatin forms a condensed structure, called a ‘chromo-
3.3.2 Chromosome Condensation Factors . . . . . . . . . . . . . . . . . . . . . . . . 36 some’, which ensures transmission of the duplicated genomic
3.4 In Vivo Mitotic Chromosome Structure . . . . . . . . . . . . . . . . . 38 DNA. The term ‘chromosome’ is derived from the Greek for
3.4.1 Inside Mitotic Chromosomes: Does the 30-nm Fibre Exist ‘coloured body’, reflecting the observation that a condensed
In Vivo? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 chromosome is clearly visible with dyes. Yet long before
3.4.2 A Novel Model of Chromosome Structure . . . . . . . . . . . . . . . . . 39
3.4.3 Organization of Genomic DNA in Interphase Nuclei . . . . . 40 the discovery of DNA as a genetic material, the mitotic
3.4.4 Dynamic Nature of the Melted Polymer . . . . . . . . . . . . . . . . . . . 41 chromosome fascinated biologists as being a candidate struc-
3.4.5 Added Note . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 ture involved in heredity. The basic mitotic chromosome
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 structure is well conserved among eukaryotes, with some
minor differences. In this chapter, we will focus on the
mitotic chromosome structure in mammals with a historical
background. We will also highlight the structural differences
between mammalian and plant chromosomes where data are
available.

3.2 Chromatin Structure

3.2.1 Nucleosome Structure

DNA has a negatively charged phosphate backbone that

produces electrostatic repulsion between adjacent DNA
regions, making it difficult for DNA to fold upon itself
(Bloomfield 1996; Yoshikawa and Yoshikawa 2002). For the
first level of folding, the long negatively charged DNA is
wrapped around a basic protein complex called a core histone
octamer, which consists of two sets of histones, H2A and H2B,
K. Maeshima (*) and H3 and H4 proteins, and forms a nucleosome (Fig. 3.1)
Biological Macromolecules Laboratory, Structural Biology Center, (Kornberg and Lorch 1999). The structural details of the
National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
nucleosome core are now known at a resolution of 1.9 Å
Department of Genetics, School of Life Science, Graduate University (Davey et al. 2002). In the core particle, 147 base pairs (bp)
for Advanced Studies (Sokendai), Mishima, Shizuoka, Japan
e-mail: kmaeshim@lab.nig.ac.jp of DNA are wrapped in 1.7 left-handed superhelical turns

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 33

DOI 10.1007/978-3-7091-1160-4_3, # Springer-Verlag Wien 2013
34 H. Takata et al.

a b
DNA 2 nm

Nucleosome

11 nm N8
N7
N8 N7 N6
N4 N6 N5
N5 N4
N3
30-nm chromatin fibre? N3
N2 N2
N1 N1

? 30 nm
c d

Nucleus
Mitotic chromosome

N7 N8
700 nm N6 N5
N5
10 µm N6
N3 N3 N4
N2 N1

Fig. 3.1 A long DNA molecule with a diameter of 2 nm is wrapped
around a core histone octamer that consists of histone H2A, H2B, H3 and
H4 proteins, and it forms a nucleosome with a diameter of 11 nm. The
nucleosome has long been assumed to be folded into 30-nm chromatin
fibres (Robinson and Rhodes 2006). How the nucleosome or 30-nm fibre
is compacted into the chromatid with a diameter of approximately
0.7 mm and interphase nucleus with a diameter of approximately
10 mm, however, remains unclear. Note that such 30-nm chromatin fibres
in native chromosomes are not detected by cryo-electron microscopy
(see Sect. 3.4.1)
around the histone octamer. Each nucleosome core is
connected by approximately 200 bp of ‘linker DNA’ to make
repetitive motifs. Accordingly, the nucleosome fibre was orig-
inally described as ‘beads on a string’ (Olins and Olins 2003).
3.2.2 30-nm Chromatin Fibre
In 1976, Finch and Klug first proposed that the nucleosome,
with linker histone H1 or Mg2+ ions, was folded into “30-nm
chromatin fibres” (Fig. 3.1) (Finch and Klug 1976). In fact,
isolated nucleosomes looked like 30-nm-diameter fibres
under transmission electron microscopy (EM). In their
model called the ‘solenoid’, consecutive nucleosomes are
located next to each other in the fibre, folding into a simple
one-start helix (Figs. 3.1 and 3.2a, c). Subsequently, a second
model of the ‘two-start helix’ was proposed based on micro-
scopic observations of isolated nucleosomes (Fig. 3.2b, d)
N2
N1
One-start helix Two-start helix
(Solenoid) (Zigzag)
Fig. 3.2 Models of a 30-nm chromatin fibre. Two well-known struc-
tural models exist for 30-nm chromatin fibres: one-start helix (solenoid)
(a) and two-start helix (zigzag) (b). Positions from the first (N1) to
eighth (N8) nucleosome are labelled as in Robinson and Rhodes (2006).
(c) In the one-start helix model proposed by Robinson and Rhodes, the
30-nm chromatin fibre is an interdigitated solenoid. Essentially, a
nucleosome in the fibre interacts with its fifth and sixth neighbours.
(d) In the two-start model proposed by Richmond and co-workers
(Schalch et al. 2005), nucleosomes are essentially arranged in a zigzag
manner such that alternate nucleosomes form interacting partners. A
nucleosome in the fibre binds to the second neighbour nucleosome.
Alternate nucleosome pairs are coloured blue and orange (The images
are reproduced from Maeshima et al. (2010) with the permission of
Elsevier)
(Woodcock et al. 1984). Although some variations exist in
this model (Bassett et al. 2009), nucleosomes are arranged
essentially in a zigzag manner, such that a nucleosome in the
fibre is bound to the second neighbour but not the first one
(Fig. 3.2b, d).
In 2004, Richmond and colleagues found that their
cross-linking study on nucleosomal arrays (12-nucleosome
repeats) was in good agreement with the zigzag conforma-
tion of the two-start helix (Dorigo et al. 2004). Furthermore,
they succeeded in resolving the crystal structure of a tetra-
nucleosome (four nucleosome cores) at a resolution of 9 Å
3 The Organization of Genomic DNA in Mitotic Chromosomes: A Novel View
(Fig. 3.2d) (Schalch et al. 2005). Although the structure
resolution is relatively low, they defined the positions of
the linker DNA and nucleosomes in the fibre by replacing
the coarse core region with the fine atomic structure of a
nucleosome core particle (Davey et al. 2002). Their results
support the zigzag two-start helix model of the 30-nm chro-
matin fibre (Fig. 3.2d).
However, more recently, Rhodes et al. reconstituted long,
regular chromatin fibres from a range of nucleosomal repeats
with chicken erythrocyte linker histone H5 (Robinson and
Rhodes 2006; Robinson et al. 2006). The in vitro assembled
chromatin fibres were observed using cryo-electron micros-
copy (cryo-EM), which is based on vitrification (frozen
hydrated) by rapid cooling, which ensures immobilisation
of all molecules in a sample to be in a close-to-native state
(for a review, see Frank 2006). Using the obtained data, they
proposed that the 30-nm chromatin fibre is an interdigitated
solenoid (Fig. 3.2a, c); their model shows that a nucleosome
in the fibre contacts with the fifth and sixth neighbours along
the DNA path (Fig. 3.2a, c) (Robinson et al. 2006), whereas
it interacts with its first neighbour in the classic solenoid
(Fig. 3.1) (Finch and Klug 1976) . Moreover, recent single-
molecule studies of long nucleosome arrays to probe their
mechanical properties also support the one-start helix topol-
ogy underlying the 30-nm fibre (Kruithof et al. 2009).
What caused this difference? Further work from Rhodes’
group suggested that the solenoid or zigzag mode of com-
paction is defined by the length of the nucleosomal linker
DNA (Routh et al. 2008). Recently, Grigoryev et al. showed
that the two-start zigzag and one-start solenoid modes may
be present simultaneously in a 30-nm chromatin fibre under
certain conditions (Grigoryev et al. 2009). Consequently, the
structural details of the 30-nm chromatin fibre remain
controversial. Nonetheless, the nucleosome chain has long
been assumed to form a 30-nm chromatin fibre before the
higher-order organization of mitotic chromosomes and also
interphase nuclei.
3.3 Structure of Mitotic Chromosomes
3.3.1 Classical Views
In the late 1970s, Laemmli and colleagues proposed a hypoth-
esis stating that chromosome structure arises from a set of
non-histone proteins that fold the chromatin fibres into loops.
They isolated histone-depleted chromosomes (Fig. 3.3a)
(Paulson and Laemmli 1977) by gently removing the
histones from isolated mitotic chromosomes via competition
with an excess of the polyanions dextran sulphate and heparin.
This approach was effective for separating the structural
contributions of the histones from those of certain non-histone
proteins. After removing the histones, they found that the
35
a
Histone
Removal
Scaffold
b
500-750 nm
200-250 nm
100 nm
30 nm
Fig. 3.3 Classical models of mitotic chromosome structure. (a) Sche-
matic representation of a histone-depleted chromosome. The histones were
removed from the isolated mitotic chromosome gently via competition
with an excess of polyanionic dextran sulphate and heparin. After removing
the histones, the DNA remained highly organized by the chromosome
scaffold (drawn in red), maintaining the size and shape of the original
chromosomes. (b) Hierarchical helical folding model. This model assumes
that 30-nm chromatin fibres are folded into 100-nm fibres and then
progressively into 200–250 nm fibres that coil to form the final mitotic
chromosomes
DNA remained highly organized by a residue of non-histone
proteins whose structure retained the size and shape of
the original chromosomes (Fig. 3.3a) (Paulson and Laemmli
1977). Therefore, Laemmli called the central axial structure
made from these non-histone proteins the ‘chromosome
scaffold’ (Fig. 3.3a). The scaffold consists of a subset of
non-histone proteins that includes two major high-molecular-
weight proteins, Sc1 (170 kDa) and Sc2 (135 kDa), and several
minor proteins (Lewis and Laemmli 1982). Sc1 was later
identified as topoisomerase II (Earnshaw et al. 1985; Gasser
et al. 1986), an evolutionary conserved protein that can
untangle DNA and relax the interwound supercoils in a DNA
molecule by passing one DNA molecule through a transient
double-stranded break in another (Wang 2002). The require-
ment of topoisomerase II for chromosome condensation
was clearly demonstrated by using fission yeast genetics
(Uemura et al. 1987) and an in vitro system in which
chromosomes were assembled by adding sperm nuclei to
extracts prepared from Xenopus laevis eggs (Adachi et al.
1991; Hirano and Mitchison 1993).
36
Such chromatin loop observations were subsequently
obtained from several EM studies (Marsden and Laemmli
1979; Earnshaw and Laemmli 1983; Maeshima et al. 2005).
As we describe later, isolated chromosomes become swollen
in low-salt buffer. Cross-sectional images of the swollen
chromosomes appear star-like; the fibres converge on the
central axis, suggesting tethering of the chromatin fibre to
the axial element, forming loops (Marsden and Laemmli
1979; Earnshaw and Laemmli 1983; Maeshima et al.
2005). This loop structure of mitotic chromosomes may
be analogous to that of the lampbrush chromosomes in
amphibian oocytes (Morgan 2002) or the meiotic prophase
chromosomes in various organisms (Kleckner 2006) that are
organized into an enormous number of large chromatin
loops emanating from a linear chromosome axis. Evidence
clearly implies that chromatin loops are a fundamental
organizing unit of chromosomes.
In 1978, another classical model, the so-called ‘hierarchi-
cal helical folding model’, was proposed from extensive
observations using high-voltage electron microscopes
(Sedat and Manuelidis 1978). This model postulates that the
30-nm chromatin fibres are folded into 100-nm fibres and
then progressively into 200- to 250-nm fibres that coil to
form the final mitotic chromosomes (Fig. 3.3b), i.e., a hierar-
chy in the chromosomes based on regular helical structures.
Indeed, EM observations showed that chromosomes appeared
to be assembled from chromatin fibres of various diameters
(Belmont et al. 1987). Furthermore, analysis using engineered
chromosomes with large copy numbers of lac operator
repeats revealed 250-nm-diameter coiling domains in the
chromosome, supporting this model (Strukov et al. 2003).
Although these two models appear incompatible, both
structures may co-exist in chromosomes. Chromosomes
often become X-shaped with helically-folded chromatids
after prolonged treatment with microtubule-depolymerising
drugs such as nocodazol or colcemid (Boy de la Tour
and Laemmli 1988; Maeshima and Laemmli 2003). Such
chromosomes show helical coiling of a fibre that is com-
posed of radial loops (Rattner and Lin 1985).
3.3.2 Chromosome Condensation Factors
3.3.2.1 Discovery of Condensins
In 1994, three groups independently made a landmark discov-
ery. Hirano and Mitchison (1994) identified a series of chro-
mosome associated polypeptides (CAPs) in Xenopus egg
extracts. Of these, the two abundant proteins, CAP-C and
CAP-E, had sequence similarity with a family of proteins in
budding yeast, which was later called the ‘structural mainte-
nance of chromosomes’ (SMC) family (Strunnikov et al.
1995). Almost simultaneously, Earnshaw’s group showed
that a major chromosome scaffold component, Sc2, is a
chicken homolog of a SMC protein (Saitoh et al. 1994).
H. Takata et al.
Yanagida’s group also demonstrated that fission yeast had
two SMC homologs Cut3 and Cut14, and that both were
involved in chromosome condensation (Saka et al. 1994).
Subsequent characterisation of the CAPs led Hirano’s
group to the discovery of a protein complex called condensin,
which consists of five different subunits, including a hetero-
dimer of CAP-C (SMC4) and CAP-E (SMC2) (Hirano et al.
1997). When condensin is depleted from Xenopus extracts
using a specific antibody, mitotic chromosome condensation
is defective, and chromatin forms swollen puffs, not a com-
pact structure. When purified condensin complex is added
back to the depleted extracts, chromosome condensation
recovers, implying that the complex has a key role in the
chromosome condensation process.
The condensin family was discovered to be conserved
from bacteria to mammals (Nasmyth and Haering 2005;
Hirano 2006), suggesting a universal role of this family in
compacting the genome. Although exactly how the complex
functions in the condensation process is unknown, interest-
ing clues came from the discovery that condensin can intro-
duce positive supercoils into closed circular DNA (Kimura
and Hirano 1997), depending on ATP-hydrolysis and
mitosis-specific phosphorylation of the complex (Kimura
et al. 1998; Takemoto et al. 2004).
Further biochemical and cytological analyses of the
chromosome scaffold demonstrated that it is composed
predominantly of a condensin complex and topoisomerase
IIa (Maeshima and Laemmli 2003). Both components have
an axial distribution with a diameter of about 200 nm at the
centre of each chromatid in swollen and compact chromo-
somes ( Saitoh et al. 1994; Coelho et al. 2003; Maeshima and
Laemmli 2003; Kireeva et al. 2004), and even in the
chromosomes of living cells (Tavormina et al. 2002; Hirota
et al. 2004). Although an argument was made that the scaf-
fold structure was an artefact resulting from the non-specific
aggregation of non-histone proteins in the histone-depleted
chromosomes (Okada and Comings 1980), these findings
support the physiological relevance of the chromosome scaf-
fold structure (Maeshima and Laemmli 2003; Maeshima and
Eltsov 2008).
Ten years after the condensin discovery in vertebrates,
condensin II was isolated as a second condensin complex,
composed of the common heterodimer of SMC2 and SMC4
and three non-SMC subunits related to, but distinct from,
those in condensin I (Ono et al. 2003; Yeong et al. 2003).
Condensins I and II show an axial localization in chromo-
somes in a rather complementary manner (Ono et al. 2003).
Experiments knocking down condensin I or II separately or
together have revealed their distinct functions in part (Hirota
et al. 2004; Ono et al. 2004). Condensin I is localized in the
cytoplasm until the nuclear envelope breaks down, whereas
condensin II is located in the nuclei during interphase.
Consistent with this observation, depletion of condensin
I does not affect prophase chromosome condensation
3 The Organization of Genomic DNA in Mitotic Chromosomes: A Novel View
(Hirota et al. 2004). By contrast, condensin II knockdown
significantly delays the initiation of prophase chromosome
condensation (Hirota et al. 2004). The chromosomal binding
of condensin II but not condensin I seems to be regulated by
phosphatase PP2A (Takemoto et al. 2009).
In the plant Arabidopsis thaliana, there are two SMC2
genes (AtCAP-E1 and AtCAP-E2) (Liu Cm et al. 2002;
Siddiqui et al. 2003) and one SMC4 gene (AtCAP-C).
AtCAP-E1 comprises more than 85 % of the total
SMC2 transcripts. The non-SMC subunits (AtCAP-H and
AtCAP-H2) of condensin I and II have also been identified
in A. thaliana (Fujimoto et al. 2005). AtCAP-H and
AtCAP-H2 are localized on mitotic chromosomes from
prometaphase to telophase; however, their localizations dur-
ing interphase are different, as in mammalian cells. While
AtCAP-H is localized in the cytoplasm, AtCAP-H2 resides
in the nucleus, particularly at the nucleolus. These different
localizations during interphase are indicative of functional
differentiation between condensins I and II in A. thaliana.
3.3.2.2 A Paradox Involving Condensin
and Topoisomerase II
No doubt exists of the universal roles of condensin and topo-
isomerase II in genome compaction. However, available
genetic and RNAi data from various organisms (Steffensen
et al. 2001; Hagstrom et al. 2002; Hudson et al. 2003) indicate
that condensin mutants mainly have a segregation defect that
does not cause dramatic abnormalities in chromosome mor-
phology. Earnshaw and colleagues showed that metaphase
chromosomes in condensin-knockout chicken DT40 cells
could still condense normally, as in wild-type cells, although
there were frequent chromosomal bridges during anaphase
(Vagnarelli et al. 2006). Thus, they inferred a novel chromo-
some condensation component, regulator of chromosome
architecture (RCA), which enabled metaphase chromosome
condensation without condensins (Vagnarelli et al. 2006).
These results suggest that condensins are required for chromo-
some structural integrity but not for chromosome condensa-
tion. As well as condensins, knockdown of the topoisomerase
II in fly or human cells also results in chromosome segregation
defects, but no prominent condensation defect ( Chang et al.
2003; Carpenter and Porter 2004; Sakaguchi and Kikuchi
2004), whereas it is essential for chromosome condensation
in some other systems (Uemura et al. 1987; Adachi et al. 1991;
Hirano and Mitchison 1993). In higher eukaryotic organisms,
which have more complex systems, proteins might acquire
more specialized functions or functional redundancy to ensure
their long-term survival, although one hypothesis states that a
residual condensin or topoisomerase II after their depletion
could still create the chromosomal shape.
In plants there is also no direct evidence so far to
indicate that plant condensins are involved in chromosome
condensation. Embryo lethality caused by the double
mutation of SMC2 genes suggests that plant condensins
37
are involved in A. thaliana development. A mutant of a
SMC4 homolog, AtCAP-C, displays phenotypes similar to
those exhibited by the AtCAP-E double mutant, including
altered segregation of alleles during gametogenesis and
embryo lethality (Siddiqui et al. 2006). Moreover, knock-
out plants of two condensin II subunits, AtCAP-G2 and
AtCAP-H2, have no defects in chromosome condensation
(Sakamoto et al. 2011).
3.3.2.3 Other Non-Histone Chromosomal Proteins
Little information is available regarding the global protein
composition of mitotic chromosomes, except for major
non-histone chromosomal proteins such as topoisomerase
II and condensin proteins. Several attempts have been
made to identify non-histone chromosomal proteins using a
proteomic approach. Some chromosome passenger proteins
required for mitotic chromosome alignment and segregation,
and also proteins located at the chromosome periphery, have
been identified (Morrison et al. 2002; Uchiyama et al. 2005;
Takata et al. 2007). The number of identified proteins
including uncharacterised ones is more than 200. Addition-
ally, several proteins specifically located at centromeric and
telomeric regions have also been reported (Foltz et al. 2006;
Okada et al. 2006; Dejardin and Kingston 2009). These
studies suggest that complex protein networks might exist
that organize mitotic chromosome structure, which might
explain why depletion of condensin or topoisomerase II
mainly results in a defect in chromosome segregation but
not condensation, as described above.
3.3.2.4 Histone Phosphorylation
Histone tails are subjected to various post-translational
modifications and could be related to chromatin structure
and function. The most striking modification of histone
tails in mitosis is the histone H3 phosphorylation at serine
10 (Guo et al. 1995) and at serine 28 (Goto et al. 1999).
A causal relationship between histone H3 phosphorylation
and chromosome condensation has been identified in Tetra-
hymena (Wei et al. 1999). A mutant in which histone H3
cannot be phosphorylated exhibits abnormal chromosome
condensation and segregation, demonstrating that phosphor-
ylation of histone H3, at least, at serine 10 is required for
proper chromosome dynamics. However, histone H3 phos-
phorylation is not required for chromosome condensation in
many other species, such as yeast (Hsu et al. 2000), Xenopus
(MacCallum et al. 2002), Drosophila (Adams et al. 2001)
and mammals (Van Hooser et al. 1998). In plants, several
studies have suggested that histone H3 phosphorylation is
involved in sister chromatid cohesion but not in chromo-
some condensation (Gernand et al. 2003). Moreover,
histone H3 phosphorylation in plant chromosomes is
strictly limited to centromeric regions in contrast to the
chromosome-wide distribution in mammalian chromosomes
(Kurihara et al. 2006).
38
3.3.2.5 Cations
Other than protein factors, such as condensins or topoisom-
erase II, cations seem to be essential participants in chromo-
some condensation. As mentioned above, mitotic
chromosomes become very swollen following the depletion
of Ca2+ or Mg2+ and this process is completely reversible.
Almost 40 years ago, Cole demonstrated that the repeated
removal and addition of Mg2+ results in cycles of chromo-
some swelling and compaction (Cole 1967). DNA has a
negatively charged phosphate backbone that produces elec-
trostatic repulsion. In the presence of cations, DNA conden-
sation results from charge neutralisation, as the binding of
cations specifically to the DNA phosphates decreases the
overall electrostatic repulsion between adjacent DNA
regions (Bloomfield 1996; Yoshikawa and Yoshikawa
2002). In the case of chromatin, the negative DNA charges
are about 60 % neutralised by core histones, which have tails
with positively charged lysine and arginine residues (Strick
et al. 2001). Therefore, the remaining approximately 40 % of
the DNA charge must be neutralised by other factors such as
histone H1, non-histone proteins and cations. In fact, using
secondary ion mass spectrometry, Strick et al. reported that
Ca2+, Mg2+, Na+ and K+ are highly enriched in mitotic
chromosomes, compared with interphase nuclei, suggesting
a potentially important role in chromosome condensation
(Strick et al. 2001). Chromatin ‘opening’ and ‘closing’
with DNA-charge neutralisation may also be involved in
global gene regulation as a result of histone tail
modifications, such as phosphorylation and acetylation
(Kornberg and Lorch 1999).
3.3.2.6 Repetitive Sequences
As more ‘complete’ genome sequences become available it
seems clear that many genomes consist mainly of repetitive
DNA sequences. For example, in humans satellite DNA
repeats are clustered in discrete areas, such as the centro-
mere, whereas other repeats such as short interspersed
nuclear elements (SINEs) and long interspersed nuclear
elements (LINEs) are dispersed throughout the genome.
Plant genomes are also composed predominantly of repeti-
tive DNA although retrotransposons with long terminal
repeats (LTR retrotransposons) are generally the predomi-
nant repeat type (see Kejnovsky et al. 2012). Are these really
“junk”? Do they have any function? One of proposed
functions is that repetitive sequences could be involved in
genome compaction. Indeed, the genomic regions, which are
abundant with satellite DNA, are condensed throughout the
cell cycle (e.g. centromere). Double-stranded DNA has the
property of self-assembly in aqueous solutions containing
physiological concentrations of divalent cations. Ohyama
and his colleagues found that DNA molecules preferentially
interact with molecules with an identical sequence and
H. Takata et al.
length, even in a solution containing heterogeneous DNA
species (Inoue et al. 2007). Therefore, postulating that
this attractive force due to repetitive sequences functions
in chromosome condensation is tempting. Furthermore,
evidence implicates LINEs (L1s) in X chromosome conden-
sation (Lyon 2003). One of the female X chromosomes is
well known to be highly condensed as a Barr body (Lyon
2003). Bailey et al. showed that the level of LINEs in X
chromosomal DNA was roughly double that in autosomes
(Bailey et al. 2000). They also found that the level was
highest in a region near the X-inactivation centre and lowest
in a short arm region containing numerous genes that escape
inactivation. These data are consistent with the postulate that
the density of LINEs correlates with the extent of inactiva-
tion or condensation. Moreover, LINEs are concentrated
mainly in the dark G-bands in autosomes, where chromatin
is condensed as heterochromatin.
‘Knob’, a classic cytogenetic marker of plant chromo-
somes, has a chromosome structure that can be identified
microscopically as being darkly stained compared to
the surrounding region. The knob was first reported in maize
(McClintock 1930). Such a lump-like structure in maize
chromosomes is a large block of heterochromatin
that contains largely tandem repetitive sequences and
retrotransposons with very few transcribed genes. Maize
knobs are mainly composed of a 180 bp repeat and a 350 bp
repeat, which are organized in tandem arrays (Peacock et al.
1981; Ananiev et al. 1998). The knob satellite is found near
the ends of all or nearly all maize chromosomes (Lamb et al.
2007). The structure of tandem repeats in such a knob could
be associated with heterochromatic domains. At the very
least, structural conservation demonstrates the importance of
tandem repeats of repetitive sequences in heterochromatin
formation, suggesting that repetitive sequences influence
chromosome condensation. Although chromosomes cannot
be condensed without the help of protein factors such as
condensins, the large amounts of repetitive sequences may
well contribute to genome compaction and have been under
selection during evolution.
3.4 In Vivo Mitotic Chromosome Structure
In the classical EM views, chromosomes seem to consist of
radial 30-nm chromatin loops that are somehow tethered
centrally by scaffold proteins such as condensin or topo-
isomerase II. Although this is a rather classical view of
chromosome organization, one must keep in mind that the
samples are fixed chemically, dehydrated and embedded in
plastic. In this section, we will introduce a recent view of
chromosome structure based on observations made in nearly
intact cell conditions.
3 The Organization of Genomic DNA in Mitotic Chromosomes: A Novel View
Fig. 3.4 (a) Cryo-electron micrograph image of a vitrified (frozen-
hydrated) section of mitotic HeLa cells. The compact areas, outlined by
the dashed line, are cross sections of mitotic chromosomes (Xs) that are
surrounded by a cytoplasm full of electron-dense ribosomes and other
particles. The scale bar indicates 500 nm. (b) Magnification of selected
3.4.1 Inside Mitotic Chromosomes: Does
the 30-nm Fibre Exist In Vivo?
What do mitotic chromosomes look like in living cells? One
of the best ways to address this question is by using cryo-EM
of vitreous sections (CEMOVIS). In CEMOVIS, after the
frozen hydrated (vitrified) cells are sectioned, the thin
sections are observed directly under a cryo-EM with no
chemical fixation or staining. This approach enables direct
high-resolution imaging of cell structures in a close-to-
native state.
More than 20 years ago, Dubochet and his colleagues first
observed vitrified sections of mammalian mitotic cells using
CEMOVIS (McDowall et al. 1986). Unexpectedly, they
found that the chromosomes showed a homogeneous and
grainy texture with a c. 11 nm spacing. Neither higher-order
nor periodic structures, including 30-nm chromatin fibres,
were recognised. Consequently, Dubochet et al. proposed
that the basic structure of the chromosome was a liquid-like
compact aggregation of 11 nm nucleosome fibres (Dubochet
et al. 1988). We also investigated human mitotic cells using
CEMOVIS and a subsequent image processing analysis and
concluded that the 30-nm fibres are essentially absent in
human mitotic chromosomes (Fig. 3.4) (Eltsov et al. 2008).
These observations are consistent with a report on the
three-dimensional structure of Xenopus chromosomes
assembled in vitro using electron tomography (K€ onig et al.
2007). In the cryo-substituted chromosomes, K€ onig et al.
detected no continuous fibre-like structures, such as 30-nm
fibres. No other regular ultrastructural organization was
observed. This conclusion is supported by the finding that
chromosome assembly in Xenopus extracts proceeds without
the linker histone H1, which supposedly stabilises the 30-nm
fibres (Ohsumi et al. 1993). Furthermore, histone H1 is
highly mobile in the chromosomes of live cells (Chen et al.
39
area of (a). Note the grainy, homogeneous texture of the chromosome
(Xs). No higher-order or periodic structures, such as 30-nm chromatin
fibres, are recognised. The scale bar indicates 100 nm (The images are
reproduced from Maeshima and Eltsov (2008) with the permission of
Oxford University Press)
2005). These data again support the absence of ‘static’
continuous 30-nm chromatin fibres in native chromosomes.
However, why can we sometimes see the 30-nm chroma-
tin fibres? As described above, the original concept of the
30-nm chromatin fibre was derived from studies using
conventional transmission or scanning EM: fibres of approx-
imately 30 nm in diameter were observed under certain
conditions involving isolated nucleosomes, nuclei and
mitotic chromosomes. To answer this question, one should
emphasise that the observation of 30-nm chromatin fibres
required a strict cationic environment, namely a low-salt
buffer containing 1–2 mM Mg2+; under such conditions,
isolated nuclei or chromosomes become swollen. Accord-
ingly, the local nucleosome concentration decreases,
favouring intra-fibre nucleosome associations, leading to
the formation of 30-nm chromatin fibres (Fig. 3.5a, b).
Furthermore, in conventional EM observations, the for-
mation of 30-nm chromatin fibres might be stabilised
through chemical cross-linking (e.g. glutaraldehyde fixation)
of intra-fibre nucleosome associations and further shrink-
age with alcohol dehydration during sample preparation
(Belmont 2006; Maeshima and Eltsov 2008).
However, at the high nucleosome concentrations that
occur in vivo, inter-fibre nucleosome interactions become
increasingly dominant (Fig. 3.5c, d). Nucleosome fibres are
forced to interdigitate with one another. This interferes with
the formation and maintenance of the 30-nm chromatin
fibre, leading to the ‘polymer melt’ state (Fig. 3.5c, d).
3.4.2 A Novel Model of Chromosome Structure
By its nature, chromatin forms aggregates in the presence of
proper concentrations of divalent or multivalent cations
(de Frutos et al. 2001). Again, how is long genomic DNA
40
Diluted chromatins
a b
30-nm folding
Chromosome e Nucleus
Fig. 3.5 (a, b) Under the diluted condition, the flexible nucleosome
fibres compact through selective close-neighbour associations, forming
the 30-nm chromatin fibres. An increase in nucleosome concentration
results in inter-fibre nucleosomal contacts, which interfere with the
intra-fibre bonds (c). The nucleosomes of adjacent fibres interdigitate
and intermix. This disrupts the 30-nm folding, and the nucleosomal
fibres progress to a state of ‘polymer melt’ (d). (e) The concept of
packaged into compact mitotic chromosomes? When
isolated chromosomes become swollen under low-salt
conditions, they seem to consist of radial chromatin loops
that are somehow tethered centrally by scaffold proteins.
Considering a structural analogy with meiotic and
lampbrush chromosomes, chromatin loops may be the fun-
damental organizing unit of chromosomes. In our cryo-EM
observations, chromosomes show a homogenous, grainy
texture with no higher-order or periodic structures
(Fig. 3.4a, b) (McDowall et al. 1986; Eltsov et al. 2008).
As the intracellular cations increase during mitosis, the loop
structures would fold irregularly toward the chromosome
centre, which contains abundant condensins. This loop
collapse process might be enhanced by the attractive force
of repetitive sequences dispersed throughout the genome.
Therefore, the compact native chromosome would be made
up primarily of an irregular chromatin network further
cross-linked by condensins (Fig. 3.6).
3.4.3 Organization of Genomic DNA in
Interphase Nuclei
The compartmentalisation of DNA within the nucleus accom-
panies genome organization. A long time ago, Rabl suggested
that interphase nuclei have a preferentially polarised organiza-
tion of centromeres at the apical side and telomeres at the basal
side in salamander nuclei (Rabl 1885). This ‘Rabl organiza-
tion’ was also observed in Drosophila (Wilkie et al. 1999),
onion, wheat and barley (Dong and Jiang 1998). However,
H. Takata et al.
Concentrated nucleosomes
c d
Melting
polymer melt implies dynamic polymer chains; i.e., nucleosome fibres
may be moving and rearranging constantly. This may provide several
advantages during chromosome condensation and segregation of
mitosis and the transcription and DNA replication processes during
interphase (The images are reproduced with minor modifications from
Maeshima et al. (2010) with the permission of Elsevier)
such organization is not found in mammalian nuclei. In the
case of plants, nuclei of small genome species exhibit diffuse
chromosome territories without Rabl organization, whereas
those of large genome species have Rabl organization (Dong
and Jiang 1998; Fujimoto et al. 2005). The Rabl orientation
might be an effective way to arrange the huge chromosomes,
at least in plants.
We next focus on the genome organization in inter-
phase nuclei. Unexpectedly, interphase nuclei in most
higher-eukaryote cell types examined by cryo-EM do not
contain a regular 30-nm chromatin fibre as the underlying
structure (Dubochet et al. 1988; Bouchet-Marquis et al. 2006;
Fakan and van Driel 2007). As Woodcock has stated (Wood-
cock 1994), this phenomenon could not be due to insufficient
contrast of cryo-EM because 30-nm fibres are not visible in
the majority of interphase nuclei after conventional chemical
fixation and plastic embedding (Horowitz et al. 1990).
Although cryo-EM analysis of specific chromatin domains,
for example, chromocentres, inactive X chromosomes and
senescence-associated heterochromatin foci, have not been
reported, typical heterochromatin regions in plant or mam-
malian nuclei that have been visualized by cryo-EM look
very similar to mitotic chromosomes (Bouchet-Marquis et al.
2006; Fakan and van Driel 2007). Consequently, the melt
may represent the predominant state of compacted
chromatin in vivo. Consistent with this, using a combination
of chromosome conformation capture (3C) technique and
polymer modelling, Dekker found that chromatin in a
transcriptionally-active domain in yeast did not form a com-
pact 30-nm fibre, but instead noted that the chromatin was
3 The Organization of Genomic DNA in Mitotic Chromosomes: A Novel View
Fig. 3.6 Novel structural model of mitotic chromosomes based on the
polymer melt concept. We postulate that chromatin loops are the
fundamental organizing unit of chromosomes. We propose that with
increased nucleosome concentration, presumably due to increases in
extended with a rather loose arrangement of nucleosomes
(Dekker 2008). The chromatin in the neighbouring domain
was more compact, but the mass density was still well below
that of a canonical 30-nm fibre (Dekker 2008).
3.4.4 Dynamic Nature of the Melted Polymer
The concept of polymer melt implies dynamic polymer
chains: nucleosome fibres may be constantly moving and
rearranging at the local level (Fig. 3.5e). During mitosis,
this dynamic irregular folding of nucleosome fibres could
be a driving force for chromosome condensation and seg-
regation. Recently, the Jun group proposed that the entro-
pic force of a dynamic flexible polymer chain can drive
bacterial chromosome segregation (Jun and Wright 2010).
Because folding of the nucleosome fibre determines DNA
accessibility (Tremethick 2007), these dynamics may have
several advantages in a template-directed biological pro-
cess, i.e., transcriptional regulation and DNA replication in
interphase nuclei. In the case of transcriptional regulation,
the dynamic movement of nucleosome fibres will contrib-
ute to the targeting of transcription factors to the cis-
elements along DNA and the forming of complexes
because target sequences are exposed more often from
the folding structure. Moreover, dynamic irregular folding
can easily form loops, facilitating interaction between pro-
moter and enhancer sequences at the three-dimensional
level.
Conclusions
We have discussed the structural aspects of mitotic
chromosomes in mammals and plants and proposed a
41
Condensin
Nucleosome
intracellular cation concentration during mitosis, the loop structures
fold irregularly toward the chromosome centre (the blue nucleosome
fibre loop), which contains abundant condensins. Note that no continu-
ous 30-nm chromatin fibres are visible in the native chromosomes
novel model. In the model, the nucleosome fibres in the
bulk of mitotic chromosomes do not form 30-nm chroma-
tin fibres but exist in a highly disordered state, which is
locally similar to a polymer melt (Figs. 3.5 and 3.6).
A similar state exists in the majority of active interphase
nuclei. The concept of the polymer melt implies dynamic
polymer chains (Fig. 3.5e). This may have several
advantages during chromosome condensation and segre-
gation during mitosis and the transcription and DNA
replication processes during interphase.
3.4.5 Added Note
In this chapter, we proposed a novel chromosome structure
model, mainly from cryo-EM observations. Recently, using
synchrotron X-ray scattering analysis, we obtained conclu-
sive evidence that human mitotic HeLa chromosomes are
predominantly composed of irregularly folded nucleosome
fibres rather than 30-nm chromatin fibres (Nishino et al.
2012). The scattering pattern implied a fractal nature in the
chromosomes, which permits a more dynamic and flexible
genome organization. We also found a similar scattering
pattern in interphase nuclei of HeLa cells in the range up to
275 nm (Joti et al. 2012). Our findings suggest a common
structural feature in interphase and mitotic chromatin: com-
pact and irregular folding of nucleosome fibres occurs with-
out a 30-nm chromatin structure (Joti et al. 2012).
Acknowledgments We are grateful to Dr. Eltosv, Prof. Dubochet, and
Prof. Frangakis for collaboration with KM We would like to thank Prof.
Laemmli, Prof. Fukui, Prof. Yoshikawa, Dr. Uchiyama and Ms. Hihara
for exciting discussions. KM was supported by a MEXT grant-in-aid
and JST CREST. HT is a research fellow of the Japan Society for the
Promotion of Science.
42
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 Structural Organization of the Plant Nucleus:
Nuclear Envelope, Pore Complexes and 4
Nucleoskeleton

Elena Kiseleva, Jindriska Fiserova, and Martin W. Goldberg

Contents 4.1 Introduction

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.2 The Nuclear Envelope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Plants, like all other eukaryotes, contain their genome within
4.2.1 Proteins of the Plant Nuclear Envelope . . . . . . . . . . . . . . . . . . . . 46 the nuclear compartment. The purpose of this compartment is
4.2.2 Lamin-Like Proteins in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 to separate the transcriptional machinery from the sites of
4.2.3 Integral Membrane Proteins of the Inner Nuclear protein synthesis. There is therefore an impermeable barrier
Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.2.4 Proteins of the Outer Nuclear Membrane . . . . . . . . . . . . . . . . . . 47
between the nuclear and cytoplasmic compartments, the
4.2.5 Ion Channels at the Nuclear Envelope . . . . . . . . . . . . . . . . . . . . . 48 nuclear envelope (NE). However the NE is permeated with
4.2.6 Functions of the Nuclear Envelope Proteins . . . . . . . . . . . . . . . 49 large protein channels, the nuclear pore complexes (NPCs).
4.2.7 The Nuclear Envelope of Plants is Similar but Distinct Trafficking through the NPCs can therefore be used to control
from Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
gene expression at several levels. The outer and inner
4.3 Plant Nuclear Pore Complex (NPC) . . . . . . . . . . . . . . . . . . . . . 50 membranes both contain distinct and complex sets of
4.3.1 Structural Organization of the Plant and Mammalian NPC
proteins, which link to the cytoskeleton as well as the nuclear
is Similar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.3.2 Plant Nucleoporins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 interior. The nucleus also therefore has important functions
4.3.3 Nucleocytoplasmic Transport Through the NPC . . . . . . . . . . 53 in organizing both the genome and the cytoplasm. Here we
4.3.4 Cell Cycle Dynamics of the Nuclear Envelope . . . . . . . . . . . . 55 describe the architecture and dynamics of the structural
4.3.5 NPC Disassembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
components of the nucleus and discuss how the plant nucleus
4.3.6 NPC Assembly at the End of Mitosis . . . . . . . . . . . . . . . . . . . . . . 56
4.3.7 NPC Assembly During Interphase . . . . . . . . . . . . . . . . . . . . . . . . . . 56 appears to differ in important ways from animals and fungi,
4.3.8 The NPC in Plants is Similar but Distinct from Other while maintaining many similarities. First we will focus on
Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 the NE of plants and their specific protein composition,
4.4 The Plant Nucleoskeleton and Intra-nuclear structure and function compared to animal systems. We
Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 will then discuss the role of the NE as a barrier and interface
4.4.1 Intermediate Filament-Like Proteins of the Nucleoskeleton 58 between the cytoplasm and nucleoplasm, before focussing on
4.4.2 A DNA Binding Integral Membrane Protein . . . . . . . . . . . . . . . 58
4.4.3 DNA Sequences of the Nucleoskeleton and Proteins That communication across the NE and finally discussing
Bind Them . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 how the nuclear interior is structurally organized by the
4.4.4 NuMA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 nucleoskeleton. Although the proteins and functions of the
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 NE and nucleoskeleton appear to overlap, we discuss them
separately so as not to confuse the distinct functions that do
clearly exist.

4.2 The Nuclear Envelope

The eukaryotic nucleus is surrounded by a double phospho-

E. Kiseleva (*) lipid bilayer called the nuclear envelope (NE). The NE of
Laboratory of Morphology and Function of Cell Structure, Institute of plants, animals or fungi is organized in a similar manner:
Cytology and Genetics, Russian Academy of Science, Novosibirsk chemically distinct inner and outer nuclear membranes
630090, Russia
e-mail: elka@bionet.nsc.ru (INM and ONM) encircle the nucleus and enclose the

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 45

DOI 10.1007/978-3-7091-1160-4_4, # Springer-Verlag Wien 2013
46
perinuclear space between them. The fusion of the two
membranes occurs at the insertion sites of the nuclear pore
complexes (NPCs), the only gates for regulated protein
transport. Inner and outer membranes of the NE differ not
only by chemical composition of their lipid components but,
importantly, by the type and number of the associated
proteins. A particular set of proteins essentially defines the
specific structural and functional characteristics of the NEs
of different cells, tissues and species.
4.2.1 Proteins of the Plant Nuclear Envelope
In animals and yeast, a combination of approaches has led to
the identification of multiple NE proteins (for review see
Dauer and Worman 2009; Hetzer 2010). In comparison, the
plant field is less advanced and only a few NE proteins have
been identified in the last decade. The following text will
concentrate on the structure and function of plant-specific
proteins putatively linked to the inner nuclear membrane
(lamin-like proteins) (1), proteins of the inner (2) and outer
(3) nuclear membrane and ion channels (4).
4.2.2 Lamin-Like Proteins in Plants
The nuclear lamina represents a multifunctional, highly
organized protein layer closely attached to the INM in
vertebrates. While biochemical, immunological and struc-
tural evidence for a lamina-like structure in plants have been
presented (McNulty and Saunders 1992; Minguez and
Moreno Diaz de la Espina 1993; Fiserova et al. 2009), the
lack of any gene coding for a plant protein homolog of
a lamin raises the question as to which proteins make up
the detected structures. Animal lamins are ~66 kDa in size,
contain a central coil-coiled region to enable dimerisation
and N- and C-terminal globular head and tail domains. In
vertebrates, lamins are encoded by three genes, LMNA,
LMNB1 and LMNB2. The three genes encode at least
seven different polypeptides resulting from splice or post-
translational modifications (for review see Gruenbaum et al.
2003; Hetzer 2010). The nuclear lamina was shown to be
essential in many processes including chromatin organiza-
tion, transcription, NPC anchoring or communication bet-
ween the nucleus and the cytoplasm. Therefore, it is likely
that some plant-specific proteins are required in plants to
fulfil at least some of the proposed lamin roles.
To date, the best candidate for a lamin-like protein in plants
is LINC1 (Little nuclei 1) in Arabidopsis alias NMCP1
(Nuclear Matrix Constituent Protein 1) in carrot (Masuda
et al. 1997; Dittmer et al. 2007). About twice the size of
lamin (LINC1 is about 134 kD), the LINC1 protein contains
a coil-coiled domain, localizes at the NE and mutations
cause altered nuclear morphology (Dittmer et al. 2007).
E. Kiseleva et al.
LINC1 belongs to a family of four LINC proteins (LINC1-4)
with varying functions and localizations within the cell.
LINC2 and LINC3 were identified as nuclear proteins with
no specificity for the NE, while LINC4 was shown to be a
plastid protein (Kleffmann et al. 2006; Dittmer et al. 2007). It
is not known, however, whether LINC1 can form filaments
in vitro or in vivo, what proteins it binds or how it is involved in
maintaining nuclear morphology.
Other putative lamin-like proteins in plants include a
family of seven filament-like proteins identified from
Arabidopsis (AtFPP1-7 for filament-like plant protein of
Arabidopsis thaliana). Homologs have been identified in
tomato (LeFPP) and rice (OsFPP) (Gindullis et al. 2002).
In yeast two-hybrid screens, these proteins associate with
another nuclear envelope protein MAF1 (MFP1 associated
factor1). However, their more specific subcellular locali-
sation or function is uncertain.
To complete the list of plant-specific coiled-coil NE
proteins, MAR binding filament-like protein 1 (MFP1) was
found to specifically bind to matrix attachment regions
(MARs) of DNA at the NE in tomato. MFP1 contains a
coil-coiled domain, an N-terminal hydrophobic region
responsible for targeting the protein to the NE but no globu-
lar head or tail. It is located at the NE and was predicted to be
a component of the plant nuclear matrix and to connect the
nuclear matrix to the NE (Gindullis and Meier 1999;
Gindullis et al. 1999).
4.2.3 Integral Membrane Proteins of the Inner
Nuclear Membrane
Most INM proteins characterised in vertebrates do not have
homologs in plants (for review see Meier 2007; Meier and
Brkljacic 2009b; Graumann and Evans 2010). The only
proteins identified to date to reside at the plant INM are
two Arabidopsis SUN-domain (Spindle architecture defec-
tive 1/UNC84 homology) containing proteins (AtSun1 and
AtSun2) (Graumann et al. 2009). Smaller than animal SUN
proteins (~50 kDa vs. ~85 kDa) they contain the highly
conserved C-terminal SUN domain and share structural
features with animal and fungal SUN-domain proteins
including a functional coiled-coil domain and nuclear
localization signal. The proteins localize to the INM and
are expressed in various tissues and cell types, but most
abundantly in proliferating tissues (Graumann et al. 2009).
Similarity searches have revealed the presence of the two
SUN proteins in other plant species including Oryza sativa,
Vitis vinifera and Zea mays. In maize, five different SUN
genes were identified (Murphy et al. 2010), which fall into
two classes. Whereas ZmSun1 and ZmSun2 represent struc-
tural homologs of animal SUNs, ZmSun3-5 include a novel
structural variant of SUN-domain proteins with likely plant-
specific roles at the NE (Murphy et al. 2010).
4 Structural Organization of the Plant Nucleus: Nuclear Envelope, Pore Complexes and Nucleoskeleton 47

Fig. 4.1 Comparison of plant and animal nucleoporins and associated proteins, showing approximate relative positions within the NPC. Proteins
labelled in bold are those that appear to differ between the two kingdoms

In vertebrates, SUN proteins interact in the intramembrane
space via their SUN-domain with the KASH domain of
KASH-domain proteins (Klarsicht/ANC-1/SYNE1 homol-
ogy, also referred to as Nesprins) located at the ONM. Thus,
they form a bridge (the bridging complexes are also known as
the LINC complexes for LInker of Nucleoskeleton and Cyto-
skeleton, which should not be confused with the LINC1-4
proteins) spanning the two membranes of the NE and
physically connecting the nuclear interior with the cytoskele-
ton (for review see Starr and Fridolfsson 2010; Starr 2011).
Thus, LINC complexes connect microtubules, centrosomes,
actin filaments, or intermediate filaments to the surface of the
nucleus. They are essential in cell cycle control, nuclear
import, and apoptosis or affect global cytoskeletal organiza-
tion (reviewed in Starr 2011). Until recently, the other partner
of the LINC complex, KASH-domain proteins of the ONM,
were not identified in plants. Interestingly, WIP proteins
(Fig. 4.1), which have a role in localizing the GTPase
activating protein for the small GTPase nuclear transport
regulator, Ran, to the outer nuclear membrane, have been
shown to contain sequences similar to the KASH domain.
Most importantly, these proteins also bind to the SUN
proteins and so appear to be functionally equivalent to animal
KASH-domain proteins (Zhou et al. 2012).
4.2.4 Proteins of the Outer Nuclear Membrane
In vertebrates, a number of spectrin repeat containing nuclear
envelope proteins (Syne/Nesprins) have been identified at the
ONM (reviewed in Mellad et al. 2011). Nesprins are
characterised, among other things, by the C-terminal KASH
domain (Klarsicht/ANC-1/SYNE1 homology) that physically
links to the SUN-domain of SUN proteins in the
intramembrane space. Nesprins thus form molecular bridges
between the cytoplasm and the nucleus termed LINC
complexes (reviewed in Starr 2011). These proteins have
important roles in nuclear positioning and migration, focal
48
adhesions and cell motility. Whether WIPs perform such
functions is unknown, but it seems likely that other candidates
will also be discovered to fulfil such roles in plant-specific
environments.
On the other hand, other plant-specific ONM proteins
have been found and studied in greater detail. MFP attach-
ment factor 1 (MAF1) is a small (~16 kDa) globular NE
associated protein first identified in tomato (Gindullis et al.
1999). Like the WIPs it contains a tryptophan/proline/pro-
line (WPP) domain. The protein is conserved in plants as
other plant species including Arabidopsis, soybean and
maize contain the full MAF1 open reading frame (Gindullis
et al. 1999). The protein interacts with MFP1 (MAR binding
filament-like protein 1) which may provide anchoring to the
NE via its N-terminal hydrophobic domains. Out of the three
MAF1 homologs in Arabidopsis called WPP1-3, only the
WPP1 and WPP2 associate with the ONM and NPCs in
undifferentiated cells of the root tip (Patel et al. 2004).
During cell division, the proteins relocate to the cell plate
and RNAi leads to developmental defects in roots suggesting
a role in cytokinesis (Patel et al. 2004).
An interesting difference between plants and animals is
the way plants use a family of ONM proteins to orientate the
Ran gradient which is required for the directionality of
nuclear transport. RanGAP is a GTPase activating protein
for Ran and is essential for nuclear transport as well as cell
division. In yeast it is located in the cytosol, whereas in
animals it is also associated with the outer surface of the
NPCs. Four RanGAPs have been identified in plants to date;
two from Arabidopsis and one each from rice and alfalfa
(Pay et al. 2002). The proteins are ~60 kDa in size and their
N-terminal domain shows significant homology to the WPP
domain of plant MAF1 protein. In interphase cells,
AtRanGAP1 localizes at the NE while it is associated with
the preprophase band, spindle and phragmoplast during
mitosis. The detected localization pattern corresponds with
the postulated function of plant RanGAPs in the regulation
of nuclear import during interphase and suggests a role as a
continuous protein marker of the plant cell division plane
(Xu et al. 2008).
RanGAP is targeted to the ONM in a plant-specific
manner. Animal RanGAP targeting to the NPCs requires
interaction of its SUMOylated C-terminus with the
nucleoporin Nup358/RanBP2 (which is, interestingly, lacking
in plants), whereas AtRanGAP1 associates with the NE via its
N-terminal plant-specific WPP domain (Rose and Meier
2001; Pay et al. 2002). Two classes of WPP-domain
interacting proteins have been further identified. Firstly,
a family of three WPP-domain interacting proteins (WIPs)
of ~70 kDa in size were identified from Arabidopsis in
yeast two hybrid screens to interact with AtRanGAP1. Simi-
larity searches showed that the protein family is conserved in
many plant species including tomato (Solanum
E. Kiseleva et al.
lycopersicum), grape (Vitis vinifera), barrel medic (Medicago
truncatula), poplar (Populus deltoides), wheat (Triticum
aestivum), rice (Oryza sativa), maize (Zea mays), barley
(Hordeum vulgare), sorghum (Sorghum bicolor), sugarcane
(Saccharum officinarum), and pine (Pinus taeda) (Xu et al.
2007). The proteins contain a predicted C-terminal transmem-
brane domain and central coiled-coil domain that is necessary
and sufficient for RanGAP interaction. WIPs co-localize with
RanGAP at the ONM as shown by confocal as well as elec-
tron microscopy. Thus, WIPs have been postulated to be
plant-specific intrinsic proteins of the ONM responsible for
RanGAP NE targeting (Xu et al. 2007).
A second class of RanGAP interacting and NE associated
proteins in Arabidopsis root tip cells is constituted of two
proteins of ~90 kDa called WPP-domain interacting tail-
anchored proteins (WIT1-2) (Zhao et al. 2008). Structurally
similar to the WIPs, both proteins contain a transmembrane
domain and coiled-coil region. Apart from the homology in
the coiled-coil region, WIPs and WITs have no further
amino acid sequence similarity. Experiments showed that
RanGAP NE targeting in root tip cells, required the activity
of at least one member of each protein family whereas both
families were dispensable in other plant tissues. This
suggested a complex RanGAP NE targeting mechanism in
higher plants contrasting to both animal cells and lower
eukaryotes (Zhao et al. 2008, reviewed in Meier et al. 2010).
4.2.5 Ion Channels at the Nuclear Envelope
In animals, the NE hosts a vast number of ion channels and
transporters (Schirmer et al. 2003; Batrakou et al. 2009).
Whether these transporters reside exclusively at the NE is
not yet certain in most cases and many are likely to be
present in other cellular membranes as well (Foster et al.
2006; for review see Matzke et al. 2010). Several nuclear
membrane ion channels have been identified in plants by
nuclear patch clamping and through genetic screens
(reviewed in Matzke et al. 2010). SERCA-type calcium
ATPases were found in tomato and Medicago truncatula
and termed LCA and MCA8, respectively (Downie et al.
1998; Capoen et al. 2011). LCA is ~116 kDa and seems to
be present only as a single copy gene in tomato. Several
putative SERCA homologs have been identified in
Arabidopsis (Evans and Williams 1998). This suggests
that, similar to animals, there may be at least three SERCA
homologs in plants located at the NE or endoplasmic reticu-
lum (Downie et al. 1998; Evans and Williams 1998). The
pump locates at the ONM and likely functions to replenish
Ca2+ stores in the perinuclear space (Bunney et al. 2000;
Mazars et al. 2011). MCA8 belongs to the large family of
calcium ATPases consisting of ten members in Medicago,
only MCA8, however, is NE targeted (Capoen et al. 2011).
4 Structural Organization of the Plant Nucleus: Nuclear Envelope, Pore Complexes and Nucleoskeleton
One ortholog containing a nuclear localization sequence was
found in Lotus japonicus. Orthologs of Arabidopsis or soy-
bean however lack this nuclear localization signal (Capoen
et al. 2011). MCA8 locates on both the INM and ONM in
Medicago, which is in contrast to the nuclear calcium
ATPases in animals that appear strictly localized to the
ONM (Humbert et al. 1996; Capoen et al. 2011). The locali-
zation at both the INM and ONM suggests a role in recapture
of calcium released into the nucleus as well as reloading the
perinuclear space (Capoen et al. 2011).
In plants, most attention has been paid to the calcium
signalling during nodulation in legumes. Perinuclear Ca2+
oscillations are produced in response to root bacterial
infections and result in the development of a specialized
organ, the root nodule. Genetic screens for nodulation-
defective mutants identified several related proteins essential
for the Ca2+ oscillations: DMI1-DMI3 (Does not Make
Infections 1–3) were identified from Medicago truncatula
and shown to be required for both nodulation and
mycorrhization (Ane et al. 2004; Riely et al. 2007). DMI1
was studied in greater detail and shown to preferentially
locate at the INM. The protein is similar to the bacterial
calcium-activated potassium channel (MthK). A conserved
motif for the potassium selective filter is, however, missing
leaving open the possibility that the protein is permeable to
other ions. Its role in calcium regulation is likely when
considering the DMI1 requirement for calcium spikes in
response to rhizobium signalling and nodule development
(Peiter et al. 2007; Mazars et al. 2009). DMI1 homologs
have also been identified in other plant species including
CASTOR and POLUX from Lotus japonicus (Imaizumi-
Anraku et al. 2005; Charpentier et al. 2008) and SYMB8
(Symbiosis8) from Pisum sativum (Edwards et al. 2007).
It has been hypothesised that CASTOR is present in the
ONM and POLUX in both the ONM and INM (Charpentier
et al. 2008). Accordingly, both channels play a role in
regulating NE membrane potential (Charpentier et al. 2008).
Nuclear calcium signalling as well as the role of the NE in
calcium signalling is a recent, strongly developing field of
plant biology and a full consideration goes beyond the scope
of this chapter. Therefore, we refer the reader to recent
reviews (Matzke et al. 2009; Mazars et al. 2009, 2011).
4.2.6 Functions of the Nuclear Envelope
Proteins
The NE represents a highly organized barrier separating the
nucleoplasm from the cytoplasm. As such it allows the
segregation of the processes involving genetic material stor-
age, expression and replication from the metabolic processes
taking place in the cytoplasm. Many roles of the NE closely
relate to the roles of the nuclear lamina and proteins
49
associated with or incorporated inside the NE. In this respect
the roles of the specific proteins and the functioning of the
NE are indistinguishable. With the assistance of the nuclear
lamina, for instance, the NE provides shape, mechanical
stability and plasticity for the nucleus (Houben et al.
2007). Growing evidence also suggests roles for lamins in
maintaining the mechanical properties of the entire cell
(Broers et al. 2006; Lee et al. 2007). In addition, lamins
are essential for chromatin organization: gene-poor regions
of chromatin have been shown to localize at the periphery
whereas gene-rich regions have been located in the nuclear
interior (Shimi et al. 2008; reviewed in Cremer et al. 2004).
Finally, lamins have been implicated to play a role in the
regulation of gene expression and DNA replication (Reddy
and Singh 2008; reviewed in Kumaran et al. 2008).
Importantly, the NE anchors NPCs and thus enables the
actively regulated transport of molecules between the
nucleus and the cytoplasm (Dechat et al. 2009). Communi-
cation of the nucleus with cytoskeletal networks is further
facilitated via transmembrane proteins and intermembrane
bridges such as the LINC complex. Microtubules, actin
filaments or intermediate filaments are connected to the
surface of the nucleus and via these bridging complexes
communicate with the nuclear interior. Thus, the two distinct
compartments are in close contact (Starr 2011) and may
affect each other according to actual needs. Details of these
interactions, though, have not been described yet (Broers
et al. 2004; Lee et al. 2007).
In animals, the importance of the NE proteins in develop-
ment and differentiation is clearly demonstrated by a
fascinating set of genetically inherited diseases referred to
as nuclear envelopathies or laminopathies. These disorders
affect muscle, adipose, bone, nerve and skin cells and range
from muscular dystrophies to accelerated aging (reviewed
in Mattout et al. 2006; Dauer and Worman 2009).
The NE of plants has received far less attention in com-
parison to animals and thus, many specific roles of the plant
NE may be only guessed. However, in complex multicellular
organisms with organized large genomes like those in some
higher plants (nuclei of many plant species contain 10–100
times more chromatin than animal nuclei) (Shaw et al. 2002;
Flowers and Purugganan 2008; Leitch and Leitch 2013, this
volume), similar complex roles of the NE are very likely.
Clearly, the NE serves as a barrier between the nucleoplasm
and the cytoplasm, and apparently provides a platform for
communication in a similar manner to that of animals: via
NPCs or via proteins physically traversing the NE such as
SUNs. The limited knowledge of the protein composition of
the INM and clear differences from animal systems may
indicate that plant-specific mechanisms exist for the involve-
ment of the nuclear periphery in nuclear processes.
Limited evidence from mutant studies of NE proteins in
plants further confirms that, like animals, NE proteins
50
function in plant growth and development: mutant plants
display not only altered nuclear morphologies but also
varying developmental phenotypes including dwarfing and
leaf curling (LINC1 mutants, Dittmer et al. 2007), root
shortening and reduced numbers of lateral roots (WPP
mutants, Patel et al. 2004). However, a combination of
approaches will be necessary to confirm the NE functions
established in animals as well as to understand plant-specific
NE roles.
One plant-specific role identified for the NE is its ability to
nucleate microtubules during interphase, as well as during
cell division. Microtubules of the mitotic spindle are formed
with the aid of microtubule organizing centres (MTOCs). To
allow access of the mitotic spindle to the chromatin, different
organisms have evolved various mechanisms. These range
between the two extremes from “closed” mitosis (where the
NE stays intact and MTOCs are either a constant part of the
NE or are inserted into the NE during the entry of the cell into
mitosis, and the mitotic spindle is formed inside the nucleus)
to “open” mitosis (where the NE is completely disassembled
as the cell enters mitosis and the mitotic spindle forms from
MTOCs in the cytoplasm) (reviewed in Guttinger et al.
2009). Open mitosis is typical for higher eukaryotes includ-
ing vertebrates and plants. While centrosomes serve as the
MTOCs in vertebrates, plants do not posses any centrosomes
or other microtubule organizing structures in the cytoplasm
and, instead, the NE has the ability to organize microtubules
and act as the MTOC (Stoppin et al. 1994; Magyar et al.
2013, this volume). This plant-specific NE function remains
far from understood and only fragmented information is
available on the molecular nature of the NE-localized
MTOC. For detailed information on plant MTOCs as well
as on the dynamics of the NE during the cell cycle we refer
the interested reader to recent reviews (Rose et al. 2004;
Meier and Brkljacic 2009b; Evans et al. 2011).
4.2.7 The Nuclear Envelope of Plants is Similar
but Distinct from Animals
Structurally, the plant NE resembles that of animals or yeast.
However, its molecular composition gives rises to a common
theme when comparing plants and animals: similar in basic
characteristics but clearly distinct in others. The absence of
plant homologs of animal NE proteins like lamins, LBR
(lamin B receptor), emerin, MAN1 or Nesprins (Graumann
and Evans 2010; Hetzer 2010), the presence of plant-specific
NE proteins such as FPP or MAF (Gindullis et al. 1999,
2002; Meier and Brkljacic 2009b) and the unique NE
targeting mechanisms of RanGAP in plants and vertebrates
(Meier et al. 2008) suggest that NE structure and composi-
tion in plants and animals were selected with respect to the
specific needs of the two different kingdoms for NE
E. Kiseleva et al.
function. However, much remains to be resolved to under-
stand not only the structural details of the NE in plants but
also the functional connections of the NE to the surrounding
environment and within the cell. Thus, this field awaits yet a
further bloom of exciting discoveries.
4.3 Plant Nuclear Pore Complex (NPC)
The NPC is a specialized multiprotein complex and is the
sole gateway for macromolecular exchange between the
nucleus and cytoplasm (Brohawn et al. 2009; Hoelz et al.
2011). Much attention has been paid to the structural and
molecular details of the NPCs and their dynamics and func-
tion in animals and yeast (Hoelz et al. 2011; Kahms et al.
2011; Onischenko and Weis 2011). In contrast, until the last
decade, little was known about the plant NPC (Heese-Peck
and Raikhel 1998; Meier and Somers 2011). Here, aspects of
the functional and structural dynamics of the plant NPC are
reviewed.
4.3.1 Structural Organization of the Plant
and Mammalian NPC is Similar
The ultrastructure of higher plant NPCs was first described
during the analysis of sections by transmission electron
microscopy (TEM) (Yoo and Bayley 1967; Roberts and
Northcote 1970). Plant NPCs appeared similar to those
observed in animal cells (Feldherr 1965; Stevens and Swift
1966; Hinshaw et al. 1992; Akey and Radermacher 1993).
Detailed structural organization of individual components of
NPCs (Goldberg and Allen 1992, 1996) as well as conserva-
tion of morphology between higher and lower eukaryotes, has
been demonstrated by field emission in-lens scanning elec-
tron microscopy (feSEM) (Kiseleva et al. 1998, 2004). These
studies have shown that vertebrate NPCs have a three-layered
ring structure with the spoke ring complex sandwiched
between the nuclear and cytoplasmic rings (Fig. 4.2) (Allen
et al. 2000; Vasu and Forbes 2001). Xenopus oocyte NPCs
have a diameter of ~110 nm (Goldberg and Allen 1995, 1996;
Stoffler et al. 1999; Allen et al. 2000; Kiseleva et al. 2000)
and possess eight-fold (top view) and two-fold (side view)
symmetry (Unwin and Milligan 1982; Hinshaw et al. 1992;
Akey and Radermacher 1993). The inner spoke ring (central
aperture ~40 nm) and central transporter (which, although
controversial, appears to alter conformation during mRNP
transport) (Akey 1990; Kiseleva et al. 1996, 1998) are within
the pore. Eight spokes emanate from the inner spoke ring,
penetrate the pore membrane and join together via radial arms
in the NE lumen to form the lumenal ring. The cytoplasmic
part of the NPC contains the star ring, which is embedded in
the outer membrane. The cytoplasmic ring lies on top and is
4 Structural Organization of the Plant Nucleus: Nuclear Envelope, Pore Complexes and Nucleoskeleton 51

Fig. 4.2 Modular structural components of the NPC based on feSEM images of NEs from Xenopus and plants (Taken from Allen et al. (2000))

composed of a thin ring with eight subunits. Rod-like
particles extend into the cytoplasm and play a role in cargo
import (Panté and Aebi 1996; Rutherford et al. 1997).
A basket-like structure attached to the nucleoplasmic ring
(Goldberg and Allen 1996; Ris 1997) contains the distal
basket ring which opens and closes during mRNP export
(Kiseleva et al. 1996). Attached to the cytoplasmic and nucle-
oplasmic rings are internal filaments which have a spoke-like
appearance (Goldberg and Allen 1996; Kiseleva et al. 1998).
Recent feSEM investigations of both sides of the NE
from tobacco BY-2 cells as well as the cytoplasmic side of
the NE from onion root cells have shown that the NPCs of
flowering plants have a conserved structure which is related
to vertebrate NPCs (Fig. 4.3) (Fiserova et al. 2009). NPCs in
plants appear a little smaller (diameter ~105 nm) than in
Xenopus oocytes (diameter 110–120 nm) (Goldberg and
Allen 1996), but they are larger than in yeast (diameter
~95 nm) (Kiseleva et al. 2004), as observed by feSEM.
The plant NPC also consists of a cytoplasmic ring formed
by eight subunits with cytoplasmic filaments, the star ring
and a central particle of 24–28 nm which is smaller than
those identified in Xenopus oocytes and Chironomus sali-
vary glands (outer diameter 33–38 nm; Goldberg and Allen
1996; Kiseleva et al. 1998). Fracturing of BY-2 cells
revealed well-preserved structures of the inner nuclear
membrane showing the existence of plant NPC baskets,
similar to those of Xenopus oocytes. Plant baskets consist
of ~6 nm filaments anchored to the nucleoplasmic ring and
52
Fig. 4.3 Nuclei isolated from onion root tip (a-c) or tobacco BY-2
cells (d-f) and visualized with feSEM at low (a and d) and high (b-f)
magnification. Large endoplasmic reticulum membranes are fused to
the outer nuclear membrane (a). Many NPCs appear distributed over
the nuclear envelope in onion root tips (b) but are arranged in rows in
7 day BY-2 cultures (as indicated in e), suggesting some underlying
organization. Such an organization could be a lamina-like structure on
the nucleoplasmic side, indicated by arrows in (f), which also shows a
BY-2 NPC basket. The eight-fold rotational repeating structure of the
plant cytoplasmic NPC ring is indicated in (c)
thicker filaments (~10 nm) extending from the distal ring of
the baskets towards the nuclear interior (Fiserova et al.
2009). It was suggested that these could have structural
roles in connecting the NPCs to each other or could act as
docking sites for transport factors. It is relevant that similar
but typically distinct inter-basket filament connections
were observed in Xenopus and bird oocytes (Jarnik
and Aebi 1991; Goldberg and Allen 1992; Goldberg et al.
1997).
The NPC density over the nuclear surface varies,
depending on species, nuclear size and activity (Lim et al.
2008). It can range from 2 to 4 NPCs/mm2 in chick
erythrocytes or mammalian lymphocytes (Maul et al.
1971) to over 60 NPCs/mm2 in Acetabularia or mature
Xenopus oocytes (Gerace and Burke 1988; G€ orlich and
Kutay 1999). The mean density of NPCs in plants varies
from over 25 NPCs/mm2 in onion root cells to 40–50/mm2 in
tobacco BY-2 cells (Fiserova et al. 2009). The NPCs are not
E. Kiseleva et al.
distributed in a random manner over the nuclear surface, but
are aligned in rows (Aaronson and Blobel 1975; Goldberg
and Allen 1992; Maeshima et al. 2006). The arrangement of
NPCs in plants into compact linear rows suggests that a
lamina-like structure could play a role in the organization
of the NPCs in plants (Fiserova and Goldberg 2010) and
other eukaryotes (mammalian cells, Maeshima et al. 2006;
Caenorhabditis, Liu et al. 2000; or in Drosophila cells,
Lenz-B€ohme et al. 1997). Loss of A-type lamins in
mammals, or lamin Dmo in Drosophila correlates with
lower NPC densities or deviant NPC spatial organization
(Sullivan et al. 1999; Maeshima et al. 2006). Moreover
yeast does not have a nuclear lamina and NPCs are arranged
as mobile clusters (Belgareh and Doye 1997; Bucci and
Wente 1997; Winey et al. 1997). NPC clustering was also
noted in Caenorhabditis mutants whose lamin content was
reduced (Liu et al. 2000). Overall, feSEM together with
other approaches has revealed further information on the
structural organization of NPCs in plants and demonstrated
that the general morphology of the NPC is conserved from
yeast to humans and plants (Fig. 4.4).
4.3.2 Plant Nucleoporins
The NPC represents the largest multiprotein channel in the
cell and is a fundamental component of all eukaryotes.
Therefore information about the proteins (nucleoporins or
Nups) that it is composed of, is essential for understanding
the mechanism of molecular trafficking. Although yeast and
mammalian NPCs differ in size, their components are simi-
larly arranged. Proteome analysis of animal and yeast NPCs
demonstrated that both contain ~30 different Nups
(Cronshaw et al. 2002; Brohawn and Schwartz 2009; Elad
et al. 2009). Although recent investigations have suggested
that the plant NPC also consists of ~30 different Nups (Meier
and Brkljacic 2009a; Meier and Somers 2011), we know
much less about the transport machinery in plants than
in vertebrates and yeast. Moreover, a complete plant NPC
proteome has not been established.
In all eukaryotes, including plants, NPC proteins form
several subcomplexes. In vertebrates, three subcomplexes
have been described: the Nup62 subcomplex (Nup62,
Nup58, Nup54, and Nup45); the Nup107-160 subcomplex
(Nup160, Nup133, Nup107, Nup96, Nup75, Nup43, Nup37,
Seh1, Sec13, and ALADIN); and the Nup93 subcomplex
(Nup205, Nup188, Nup155, Nup93, and Nup35) (Tran and
Wente 2006). Other investigators have separated Nups into
three categories, depending on their location in the different
compartments within the pore membrane: Nups integrated
into the pore membrane itself; scaffold Nups involved in
the formation of the NPC framework; and barrier Nups
critical for the selective permeability of the NPC (Fig. 4.1)
4 Structural Organization of the Plant Nucleus: Nuclear Envelope, Pore Complexes and Nucleoskeleton
Fig. 4.4 Evolutionary
conservation of the structural
organization of NPCs from
different organisms, showing
NPCs from onion (a), yeast (b)
and Drosophila embryos (c)
(Onischenko and Weis 2011; Tamura and Hara-Nishimura
2011).
The first layer consists of membrane Nups that facilite the
anchoring of the neighbouring scaffold layer Nups. This
layer in turn attaches barrier Nups that form the wall of the
central channel and the asymmetric NPC extensions
(Figs. 4.1 and 4.2). Barrier Nups contain multiple
phenylalanine-glycine (FG) repeats giving rise to either a
hydrogel based on hydrophobic interactions between the FG
repeats (Ribbeck and G€ orlich 2001) or a brush border
arrangement of the intrinsically disordered FG domains
(Rout et al. 2000). The permeability properties of these
molecular organizations allow selective access of the trans-
port complexes while inhibiting the entry of other proteins of
comparable size. Nups localize on both sides of a symmetri-
cal axis in the NE plane.
A number of plant Nups that are similar to the animal and
yeast Nups have been found using large scale genetic and
reverse genetic approaches (Boruc et al. 2012). Sequence
similarity searches have failed to identify plant homologs
for about half of the nucleoporins identified in metazoan
NPCs (Xu and Meier 2008). Nevertheless, proteome analysis
of the nuclear pore composition in Arabidopsis using trans-
genic Arabidopsis expressing GFP-tagged RAE1 (RNA
export factor 1) and mass spectrometry led to the identifica-
tion of a total of 30 Arabidopsis Nups (Tamura et al. 2010).
Most of these proteins exhibited sequence homology to verte-
brate Nups however no homologs in the Arabidopsis genome
were found for the human Nup358, Nup153, Nup45, Nup37,
NDC1, or POM121 (indicated in bold in Fig. 4.1) (Xu et al.
2007; Tamura et al. 2010). A candidate for Arabidopsis
NDC1, which was localized at the NE in Arabidopsis root
tip cells, was proposed (Stavru et al. 2006). Nup136 is thought
to be a functional homolog to animal Nup153, although they
have no sequence homology (Tamura et al. 2010). Interest-
ingly overexpression of Nup136 affects nuclear morphology.
Plant Nup136 (Tamura et al. 2010), also named Nup1 (Lu
et al. 2010), is a FG-Nup.
Plant Nups are important in diverse processes such as the
resistance response and hormone signaling (Parry et al.
2006), cold-stress tolerance (Dong et al. 2006), or rhizobial
53
and fungal symbiosis (Kanamori et al. 2006; Saito et al.
2007; Boruc et al. 2012).
4.3.3 Nucleocytoplasmic Transport Through
the NPC
Small molecules and ions diffuse freely through the NPC,
whereas the transport of large molecules such as proteins and
RNA is a regulated process (W€alde and Kehlenbach 2010;
Zhao and Meier 2011). The mechanism of nucleocy-
toplasmic transport through the mammalian NPC has been
thoroughly studied. Most actively transported proteins con-
tain a NLS (nuclear localization signal) or NES (nuclear
export signal), or indeed both. These motifs have also been
found in many plant proteins (Merkle 2001). Selective trans-
port is regulated by many soluble factors. The most impor-
tant carrier proteins belong to the superfamily of importin
b-like proteins (also referred to as “karyopherins” or
importins, exportins and transportins) (Fried and Kutay
2003; Xylourgidis and Fornerod 2009). Karyopherins are
conserved eukaryotic proteins but in plants they do reveal
some unique features (Meier 2007).
Another soluble factor, the small GTPase Ran, exists in
either the GTP- or GDP-bound form. RanGTP and its con-
version to RanGDP crucially control the interactions
between karyopherins and cargoes (Merkle 2001; Meier
2007; Xu et al. 2007). Three genes encoding Ran have
been identified in Arabidopsis (Haizel et al. 1997). Transfor-
mation of RanGTP to RanGDP takes place in the cytoplasm
and is under the control of Ran GTPase Activating Protein
(RanGAP) and Ran Binding Proteins (RanBPs). The
chromatin-bound Ran nucleotide exchange factor RCC1
(regulator of chromosome condensation) converts RanGDP
to RanGTP in the nucleus. These nuclear transport factors
and the import factor for RanGDP (NTF2) maintain the
nucleocytoplasmic gradient between RanGTP within the
nucleus and RanGDP in the cytoplasm which determines
directionality of nucleocytoplasmic transport (W€alde and
Kehlenbach 2010). Most components of the Ran cycle
54
Fig. 4.5 Summary of the steps of
protein import and export in
plants. Imp import receptor, NLS
nuclear localization sequence
containing cargo protein, NES
nuclear export sequence
containing protien, exp export
receptor (for details see text)
have now been identified in different species of plants
including RanGAP, RanBPs, and two homologs of NTF2
(Xu et al. 2007; Meier and Brkljacic 2009b).
The classical import process in animals and plants starts
with the binding of proteins with NLSs to importin a which
then binds to importin b. Importin b mediates the transloca-
tion of the complex across the NPC via interaction with
FG-domains. Figure 4.5 illustrates the steps of nucleocy-
toplasmic transport of molecules across the NPC governed
by Ran. It has been suggested that FG-Nups may play a dual
role in nuclear transport: they may create a permeability
barrier for the Nups, and they may transiently interact with
the soluble nuclear transport receptors (NTRs) mediating the
translocation across the NPC. Several importin a and 17
importin b-like proteins have been identified in Arabidopsis
(Bollman et al. 2003; Meier 2007). In contrast to animals,
the plant importin At-IMPa is capable of mediating nuclear
import in the absence of importin b in in vitro experiments
(Hubner et al. 1999). Proteins transported to the nucleus
dissociate from the cargo after binding of RanGTP to
importin b, and this is followed by recycling of the importin
b-RanGTP complex back to the cytoplasm.
Proteins containing a NES interact with the nuclear export
receptor, CRM1, also known as exportin-1, which is the major
karyopherin that exports many cellular proteins from the
nucleus to the cytoplasm. The homologs of different export
karyopherins such as exportin 1 (AtXPO1), exportin 5
(HASTY) and exportin t (PAUSED, involved in tRNA export)
were determined and functionally characterized in plants
E. Kiseleva et al.
(Meier 2007). For protein export, RanGTP associates broadly
with the nuclear export receptor/cargo and this complex
translocates across the NPC to the cytoplasm.
The dissociation of the transport complex is triggered in the
cytoplasm by GTP-hydrolysis on Ran under the action of the
RanBP1 and RanGAP. This is followed by recycling of the free
exportin back to the nucleus. Export of mRNA to the cytoplasm
is controlled by different mechanisms that are not dependent on
karyopherins nor on the Ran system (Kelly and Corbett 2009).
At present there are several models that have been developed to
explain how macromolecules pass through the NPC central
channel (W€alde and Kehlenbach 2010).
Despite considerable similarities between the nuclear
transport machineries of plants and animals (reviewed in
Meier 2000; Xu et al. 2007) there are clear-cut differences
between the Ran components in cells from these two
kingdoms. The Ran-specific guanine nucleotide exchange
factor RCC1 has not so far been described in plants, although
this function must exist. Plant RanGAP1 contains a unique
WPP domain at the N-terminal end which has not been
found in vertebrates (Rose and Meier 2001). Two novel
proteins (WIP and WIT) which control RanGAP1 binding
to the NE have been described (Meier and Brkljacic 2009b).
The vital importance of plant transport factors is suggested
by studies showing that defects in their expression have
major effects on plant development (Zhao et al. 2008;
Meier and Brkljacic 2009a). In particular, the functional
role of RanGAP in plant cytokinesis has been demonstrated
(Xu et al. 2008).
4 Structural Organization of the Plant Nucleus: Nuclear Envelope, Pore Complexes and Nucleoskeleton
4.3.4 Cell Cycle Dynamics of the Nuclear
Envelope
The cell cycle in all eukaryotes is controlled by cyclin-
dependent kinases (CDKs), see Magyar et al. 2013; this volume.
Mitosis equally partitions replicated chromosomes as well as
duplicated cytoplasmic organelles into the daughter cells
(Prunuske and Ullman 2006; Costas et al. 2011). CDKs are
also central in controlling the steps of interphase (Dultz and
Ellenberg 2010; Talamas and Hetzer 2011). Cell cycle
transitions depend on protein phosphorylation and dephosphor-
ylation, which is controlled by CDKs and cyclins, as well as
phosphatases. The higher-plant cell cycle differs from other
higher eukaryotes because plants do not have centrosomes, as
discussed above. Instead the NE plays the role of the microtu-
bule organizing center (MTOC) (Stoppin et al. 1994). Addition-
ally, in contrast to metazoans, mitotic nuclear membranes
remain in close proximity to the chromosomes and NE refor-
mation is spatially organized in dividing plant cells (Graumann
and Evans 2011). Before preprophase, plant CDKs and cyclin B
move from the cytoplasm to the nucleus and accumulate at the
NE, initiating its disruption and chromosome condensation
(Boruc et al. 2012). Mitotic NE disassembly in metazoans
involves the disintegration of NPCs, lamina depolymerization
and retraction of nuclear membranes into mitotic endoplasmic
reticulum (ER). The mechanism of this well orchestrated basic
process is not completely understood (Maeshima et al. 2011).
There are three types of mitosis: open (higher plants and
animals), closed (lower eukaryotes such as yeast) and semi-
closed (e.g., Drosophila early embryos) (Kiseleva et al. 2001).
NPCs are assembled in the re-forming nuclear envelope (NE)
during the exit from mitosis as well as into intact, expanding
NEs during interphase. During open mitosis, NPCs, membranes
and the nuclear lamina are broken down in prophase and
reassembled around the daughter chromosomes in telophase
(Fernandez-Martinez and Rout 2009). During interphase, new
NPCs are embedded in the intact NE and double the NPC
number before the nucleus starts to divide. On the basis of
biochemical and genetic studies, in vitro assembly assays and
Nup dynamics during the assembly, two models for NE and
NPC reformation have been developed (Hetzer et al. 2005;
Doucet et al. 2010). One model proposes that fusion of the
outer and inner membrane creates a pore for NPC assembly.
In the other model specific Nups bind to chromatin to form “pre-
pores” and the stepwise assembly of NPC structures then
follows. At the final step, the membrane encloses newly formed
NPCs and peripheral nucleoporins are added.
4.3.5 NPC Disassembly
While we have significant information about NPC composi-
tion, our understanding of assembly and especially disassem-
bly of this multiprotein complex is not completely clear
55
(Laurell et al. 2011). In higher eukaryotes, including plants,
the NPCs disassemble in late prophase. This is controlled by
the family of evolutionary conserved CDKs, complexed with
their regulatory cyclin B and NIMA (Onischenko et al. 2005;
M€uhlh€ausser and Kutay 2007; Magyar et al. 2013, this
volume), resulting in phosphorylation of many Nups
(Macaulay et al. 1995; Laurell et al. 2011). NPC disassembly
is a quick process (about 1 min) and starts with disassembly of
the peripheral NPC components such as the cytoplasmic ring
and filaments, together with the star ring, then the central
transporter and finally the scaffold and transmembrane
nucleoporins (Kiseleva et al. 2001; Lénárt et al. 2003). In
metazoans the NE loses its integrity during the transition
from prophase to prometaphase (Guttinger et al. 2009) and
the pore membrane proteins diffuse into the ER network. In
vertebrates during mitosis the GLFG (glycine-leucine-
phenylalanine-glycine) nucleoporin Nup98 localizes to both
sides of the NPC with its partner Rae1 and the cytoplasmic
filament protein Nup358/RanBP2. Nup98 is phosphorylated
earlier than other Nups such as gp210, Nup214, Nup153, the
Nup62 subcomplex, the Nup53/93 subcomplex, and the
Nup107-160 subcomplex (Glavy et al. 2007; Dultz et al.
2008). It has been demonstrated that phosphorylation of
Nup98 by multiple kinases is crucial for NPC disassembly
during entry into mitosis (Laurell et al. 2011). Disassembly
and reassembly of the NPC during mitosis is also regulated by
a dynamic equilibrium between the activities of CDK1 and
okadaic acid-sensitive protein phosphatases (Onischenko et al.
2005). Disassembly of NPCs in plants has not been
investigated to the same extent as in animals. Mitotic protein
phosphorylation releases Nups from the plant NPCs and they
become distributed on the spindles or soluble in the cytoplasm
at late preprophase (Boruc et al. 2012). It has been shown in
plants that Nup136/Nup1 disperses into the cytosol during
NE breakdown and then reassociates with the NPCs as the
NE reforms in the daughter cells (Xu et al. 2007). NUA
(Nuclear Pore Anchor, the Arabidopsis homolog of Tpr/
Mlp1/Mlp2/Megator) and Rae1 relocate during mitosis
(Lee et al. 2009).
Because plants have no centrosomes there is a prepro-
phase period during the G2 phase of the cell cycle when the
NE plays the role of MTOC (Stoppin et al. 1994; see also
Magyar et al. 2012, this volume). Before this stage, plant
CDKs and cyclin B move from the cytoplasm to the nucleus
and accumulate at the NE initiating its disruption and chro-
mosome condensation (Boruc et al. 2012).
Apart from mediating nucleocytoplasmic transport in
higher eukaryotes, Nups play an important role in the regu-
lation of mitosis (Meier and Brkljacic 2009a). After phos-
phorylation and disassembly most animal Nups are soluble
and disperse in the cytoplasm. Some Nup complexes
(Nup62, Nup88 and Nup107) are associated with spindles
or kinetochores in metaphase. For example, it has been
found that Nup98 participates in the regulation of the
56 E. Kiseleva et al.

anaphase promoting complex during early mitosis and

spindle assembly (Laurell et al. 2011). The Nup107-160
complex is involved in mitotic microtubule (MT) assembly
and together with Nup358 in stabilization of MT/kineto-
chore attachment (Zuccolo et al. 2007). Nup358 (RanBP2),
which is located to the cytoplasmic filaments in animals,
plays an additional role in chromosome segregation.
Nup133 is required for efficient anchoring of tubulin
associated proteins, the dynein–dynactin complex, to the
NE. Tpr and Nup153 are active players in the SAC (spindle
assembly check point) regulation and Nup153 together with
Nup62 are critical for cytokinesis (Mackay et al. 2009).
Plant Nups and related factors might also play a role in
cell division and cytokinesis (Meier and Brkljacic 2009a).
It was shown that Arabidopsis NUA (a plant Nup with a
role in control of flowering time and development) can
influence gene expression (Xu and Meier 2008). Mutations
in Arabidopsis RanBP1 cause mitotic arrest in metaphase/
anaphase and cell death in the root meristematic zone
(Kim et al. 2001). Plant Nup136, which is probably a
functional homolog of animal Nup153, is involved in reg-
ulation of nuclear morphology (Tamura et al. 2010). The
quantity of Nup136 on the NE determines nuclear shape in
Arabidopsis (Tamura et al. 2010). Mutations in the plant
Nup107–160 complex lead to mitotic as well as nucleocy-
toplasmic transport defects (Xu and Meier 2008).

4.3.6 NPC Assembly at the End of Mitosis

NPC assembly at the end of mitosis requires dephosphoryla-
tion of Nups and the correct recruitment of all the dispersed
components back into the multiprotein NPC. The steps of this
process were first investigated in detail in vitro. Studies have
shown that NPCs assemble after the double nuclear membrane
forms, a process that starts with the binding of membrane
vesicles to chromatin (Lohka and Masui 1984; Salpingidou
et al. 2008). Plant NE reassembly commences in anaphase and
appears to be completed concomitant with early cell-plate
formation (Xu et al. 2007). In late anaphase these membranes
and Nups are recruited to the surface of decondensing
chromatin and rapidly form NE fragments in which NPCs
are assembled (Dultz et al. 2008). In animals, the
Nup107-160 complex is recruited first to the reforming NE
and this step is mediated by DNA-interacting Nup, ELYS
(Belgareh et al. 2001; Rasala et al. 2008). This is followed
by the incorporation of membrane vesicles containing trans-
membrane Nups, POM121 and NDC1, which in turn leads to
the incorporation of other Nups (Onischenko et al. 2009).
Nup133 and Nup62, then other Nups are recruited in a step-
wise manner relatively late in the assembly process (Bodoor
et al. 1999; Dultz et al. 2008). The sequentially formed struc-
tural intermediates of the NPC (dimples, pores, star-rings, and
Fig. 4.6 Structural intermediates visualized during NPC assembly and
the corresponding model of the steps. (a) NPC intermediates in the
nuclear envelope of a telophase nucleus isolated from Drosophila early
embryo; (b) schematic representation of the steps of NPC formation;
(c) NPC intermediates in the nuclear envelope of the nucleus isolated
from tobacco cell culture
thin rings) have been identified on growing NEs assembled in
Xenopus egg extracts (Goldberg et al. 1997), and also in the
NEs of actively dividing nuclei from early Drosophila
embryos by feSEM (Kiseleva et al. 2001). Similar NPC
intermediates have recently been demonstrated (Fig. 4.6) dur-
ing investigations of plant nuclei from proliferating tobacco
BY-2 cells (Fiserova et al. 2009). This supports the
conclusions about the conserved mechanism of NPC assembly
through intermediates in higher eukaryotes.
4.3.7 NPC Assembly During Interphase
NPC biogenesis during interphase may differ from mitosis
because NPCs must be inserted into a fully formed NE lined
by the nuclear lamina (Maul et al. 1971; Goldberg et al.
1997; Winey et al. 1997; Dultz and Ellenberg 2010; Talamas
and Hetzer 2011). The membrane curvature-inducing
4 Structural Organization of the Plant Nucleus: Nuclear Envelope, Pore Complexes and Nucleoskeleton
protein reticulon 4a (Nogo A) is involved in local fusion of
the inner and outer NE membranes at the site of the forming
pore (Kiseleva et al. 2007; Dawson et al. 2009). It has been
suggested that the lipid composition of the membrane is
critical for the early steps of NPC assembly before the
incorporation of Nups (Fichtman et al. 2010). It was shown
that POM121, rather than the Nup107–160 complex, is
recruited first to the site of the new pore and plays a role in
the early steps of NPC assembly (Funakoshi et al. 2011;
Talamas and Hetzer 2011). Sun1 is an INM-specific protein,
found in plants, animals and fungi. It interacts with the outer
membrane nesprins in animals, linking the inner membrane
to the actin network in the cytoplasm and possibly the
nucleus. In plants it binds WIP/WIT proteins, but conne-
ctions to the cytoskeleton or nucleoskeleton have not yet
been demonstrated. In animals, Sun1 is present with
POM121 at the forming NPC and may promote membrane
fusion (Talamas and Hetzer 2011). There is evidence from
vertebrates that the Ran GTPase cycle, the membrane layer
Nup53 and the DNA-binding nucleoporin, Mel28/ELYS are
necessary for the early steps of NPC biogenesis (Onischenko
and Weis 2011). Currently nothing is known about plant
NPC assembly during interphase.
4.3.8 The NPC in Plants is Similar but Distinct
from Other Organisms
Significant progress has been achieved in unravelling the
structural organization and biochemical composition of the
plant NPCs. Plant and animal NPCs are similar in many
respects supporting the notion that NPC organization is
conserved in evolution. However our knowledge is far
from complete and it is likely that not all plant Nups or
nuclear transport proteins have been discovered. Important
remaining questions are as follows: how are NPCs assem-
bled during interphase? how do Nups contribute to different
cellular functions in addition to nuclear transport? to what
extent are Nup complexes in plants and animals similar?
More detailed analysis of Nup dynamics and the role of
specific FG domains should increase our understanding of
the mechanisms of nucleoplasmic transport in plants which
may reveal some unique features. Further work should focus
on the connection between apparently plant-specific nuclear
pore components and their fate and function during plant-
specific aspects of the cell cycle.
4.4 The Plant Nucleoskeleton
and Intra-nuclear Organization
The enclosure of the genome into a compartment that
separated the transcription process from the translation
machinery was a pivotal event in the evolution of complex
57
Fig. 4.7 Thin section TEM comparing Arabidopsis thaliana root tip
cells (b) to rat gastric gland cells (a), showing the highly organized and
consistent nature of plant nuclei, like in many animal tissues
eukaryotes. It enabled an exquisite control of gene expres-
sion by regulating the access of proteins such as transcription
factors to the genome and exit of RNAs from the nucleus. As
discussed, the nuclear envelope provides the impermeable
wall to the compartment and the NPCs are the selective and
controllable gates.
The evolution of the nuclear compartment also allowed
the genome to be dynamically organized on several levels.
For instance, the location of chromosomes, chromosomal
domains and even individual genes can be controlled within
the nuclear space. The control of such localization may be
developmental, leading to epigenetic control, where house-
keeping genes and tissue-specific genes are located to active
euchromatic regions whereas genes that are not required are
located to inactive heterochromatin. Alternatively, recent
evidence from budding yeast (Taddei 2007) has shown that
gene activity can be switched on, or enhanced, by relocating
specific genes from the nuclear interior to the NPCs on a
short timescale. On top of this, higher plant genomes can be
very large, with multiple gene duplications and differentially
expressed variants as well as vestigial genes.
Plants in particular, therefore, have a requirement to orga-
nise the genome into regions of different activity which can be
controlled either during development or in response to specific
signals. It also must organize other processes such as DNA
replication and RNA processing. In addition, examination of
electron micrographs of plant tissue, as well as animals, gives
the immediate impression of a highly regulated and consistent
organization (Fig. 4.7).
58
How this organization is achieved is not fully understood,
especially in plants. A basic requirement for a spatial
organizing system is a structural framework. Such a frame-
work can be used to provide a semi-rigid set of spatial
co-ordinates, onto which specific regions of the genome, or
other functional centres, can be attached in specific domains.
For instance in animals, a subset of heterochromatin is
located to the peripheral nuclear lamina (Towbin et al.
2009). This structural framework, which includes the nuclear
lamina, is known as the nuclear matrix or nucleoskeleton.
The nuclear matrix was discovered using electron micros-
copy ~35 years ago in rat liver cells (Berezney and Coffey
1974, 1977), but partly because of the highly extracted nature
of this structure it remains controversial to this day.
This latter point was partially addressed by Jackson and
Cook (1988), who observed an intermediate filament-like
network throughout the nucleus after removal of chromatin
using “physiological” buffers. This was termed the “nucleo-
skeleton” and interestingly was shown to retain enzymes
involved in transcription and DNA replication (Hozák
et al. 1993), suggesting that the framework has a direct
role in organizing biochemical processes of the nucleus.
Although the protein composition of the nucleoskeleton
has not been fully elucidated, antibodies raised against ani-
mal nuclear matrix preparations appear to react with plant
nuclei (Chaly et al. 1986). A number of studies subjected
plant cells to nuclear matrix preparation methods and
observed filamentous structures similar to the animal nuclear
matrix/nucleoskeleton (Frederick et al. 1992; McNulty and
Saunders 1992; Mı́nguez et al. 1994; Wang et al. 1996;
Blumenthal et al. 2004). Therefore it can probably be
concluded that if this is a bone fide structure in animals, it
probably also exists in plants.
4.4.1 Intermediate Filament-Like Proteins
of the Nucleoskeleton
The nucleoskeleton consists of two structurally distinct
components: the nuclear lamina and the intra-nuclear net-
work. All multicellular animal species studied so far, from
hydra to humans, possess lamins (Erber et al. 1999; Burke
and Stewart 2006; Melcer et al. 2007; Mencarelli et al.
2011). Lamins constitute the archetypal intermediate fila-
ment network that lines the nucleoplasmic face of the inner
nuclear membrane. The nuclear lamina provides the outer
structural shell to the nucleus and is clearly involved in the
structural rigidity of the nucleus (Sch€ape et al. 2009). In
humans and in animal models, certain lamin mutations result
in easily damaged nuclei (Shimi et al. 2010). Interestingly,
the study of disease causing mutations in humans has
revealed that lamins also have important roles in organizing
chromatin and the nucleus in general (Shimi et al. 2010) and
E. Kiseleva et al.
can therefore be considered as an integral, or even a major
structural and functional component of the nucleoskeleton.
Furthermore lamins are also found in intra-nuclear structures
(Markiewicz et al. 2002), and may be an integral component
of the internal nucleoskeleton. However we do not know if
internal lamins form intermediate filaments and indeed they
appear to be more easily extracted than the peripheral lamina
and other nuclear matrix proteins (Markiewicz et al. 2002).
There is both structural and immuno-histochemical evi-
dence for a nuclear lamina-like structure in plants (McNulty
and Saunders 1992; Minguez and Moreno Diaz de la Espina
1993; Fiserova et al. 2009). However, the protein composi-
tion of this structure has not been characterised. Lamins are
coiled-coil containing intermediate filament proteins and
there are coiled-coil proteins that localize to the NE (e.g.,
NMCP1, LINC1 and MFP1, as discussed above). These are
therefore candidate lamin-like proteins. However, of these,
only LINC1 is exclusively located to the NE when expressed
as a YFP-chimera (Dittmer et al. 2007). LINC2-YFP, on the
other hand, is found throughout the nucleoplasm (Dittmer
et al. 2007) and therefore could perform similar functions to
the intra-nuclear lamins or other filamentous nuclear matrix
components. Like the intra-nuclear lamins however, it is not
known if LINC2 assembles into filaments. Interestingly,
mutation of both LINC1 and LINC2 in A. thaliana results
in stunted plants with small, misshapen nuclei, reminiscent
of the “laminopathy” phenotypes in humans (Worman et al.
2009). It is likely therefore that LINC1 and LINC2, as well
as LINC3, are important constituents of the different
nucleoskeletal components: the lamina and the intra-nuclear
network respectively.
Much of the evidence for a plant nucleoskeleton comes
from staining nuclear matrix preparations with antibodies
against intermediate filaments in general and lamins in
particular (Frederick et al. 1992; McNulty and Saunders
1992; Mı́nguez et al. 1994; Wang et al. 1996; Blumenthal
et al. 2004). Usually such antibodies stain throughout the
nuclear matrix preparations. This shows that there are lamin/
intermediate filament-like epitopes throughout the plant
nucleus. Despite this, no sequences within plant genomes
have been recognised that code for any protein that conforms
to the properties of any intermediate filament protein. Plants
do possess long coiled-coil proteins (Rose et al. 2004, 2005),
but they do not have other clear intermediate filament
structural properties.
4.4.2 A DNA Binding Integral Membrane
Protein
MAR binding filament like protein 1 (MFP1) is an interest-
ing long coiled-coil protein that locates to the nuclear
periphery. It is thought to be membrane bound due to the
4 Structural Organization of the Plant Nucleus: Nuclear Envelope, Pore Complexes and Nucleoskeleton 59

predicted transmembrane domain. This is in contrast to be used to enhance their expression (Maximova et al. 2003;
lamins which become membrane associated via a lipid mod- Van der Geest et al. 2004; Wang et al. 2007). Topoisomerase
ification. MFP1 also has DNA binding activity (Meier et al. II is a component of the nuclear matrix in animals and MARs
1996) and appears to have a preference for DNA sequences contain topoisomerase II consensus sequences (Berrios et al.
known as Matrix Attachment Regions (MARs). MARs (also 1985). It is uncertain however whether plant MARs are topo-
known as scaffold attachment regions or SARs) are ~200 bp isomerase II targets. Treatment of plant nuclei with topoisom-
long AT-rich DNA sequences which are substrates for topo- erase II inhibitors, which cause double stranded breaks in the
isomerase II and are found tightly bound to nuclear matrix DNA, result in fragmentation of the genome into consistent
preparations after extensive nuclease digestion of the DNA sized fragments (Razin et al. 1991). This suggests a role for
and biochemical extraction (Wang et al. 2010). MARs there- topoisomerase II in determining chromatin domains. How-
fore are thought to anchor DNA to the nuclear matrix. ever, it is uncertain whether topoisomerase II actually cleaves
In some studies MFP1 is located in a speckled pattern to at the MARs in Arabidopsis (Makarevitch and Somers 2006).
the nuclear periphery (Gindullis and Meier 1999), but others
(Samaniego et al. 2006) indicate that it is intra-nuclear. The
latter localization would be unusual if MFP1 was truly an
4.4.4 NuMA
integral membrane protein, as predicted from the sequence,
because as far as we know there are no intra-nuclear
Another protein identified as a consistent constituent of various
membranes in plant cells. It is however possible that MFP1
nuclear matrix preparations is Nuclear Mitotic Apparatus
exists as a membrane bound or soluble protein depending on
(NuMA) (Radulescu and Cleveland 2010). Nuclear matrix
its functional state. Unfortunately the picture for MFP1 is far
preparations from onion cells contain a large protein (similar
from clear: we know little about its function and there is
to human NuMA) that is recognised by anti-NuMA antibodies
evidence that it may also be associated with chloroplasts and
(Yu et al. 1999). These antibodies also label nuclear matrix
Golgi as well as or instead of the nucleus (Jeong et al. 2003;
filaments as shown by immuno-electron microscopy. Although
Samaniego et al. 2006).
this protein shares similar properties with mammalian NuMA,
An MFP1 binding protein has been discovered called
no NuMA homolog has been clearly identified in plants. Mam-
MFP1 associated factor (MAF1) (Gindullis et al. 1999).
malian NuMA is a large coiled-coil protein that tethers
MAF1 contains a WPP (tryptophan-proline-proline) domain
microtubules to the spindle poles during mitosis but is also
which is an interaction domain also found in RanGAP. In
associated with the nuclear matrix during interphase (Kallajoki
RanGAP the WPP domain binds to WPP domain-interacting
et al. 1991). When over-expressed, intra-nuclear filaments are
proteins (WIPs) and WPP domain-interacting tail-anchored
observed (Gueth-Hallonet et al. 1998), suggesting it could
proteins (WITs) (Zhao et al. 2008; Brkljacic et al. 2009) in
constitute a structural component of the nucleoskeleton. There-
order to locate it to the nuclear envelope (Meier et al. 2008).
fore like several nucleoskeletal components, there is likely to
Recently (Zhou et al. 2012) it was shown that WIPs may in
be a functional equivalent of NuMA, which may be structurally
fact be the elusive equivalent to metazoan KASH-domain
similar enough to react with antibodies raised against the
proteins, which link the outer nuclear membrane to the
mammalian protein, but without enough sequence similarity
nuclear interior via the inner nuclear membrane SUN
to be recognised by sequence comparison algorithms.
proteins. It is possible therefore that the function of MFP1
is to locate other proteins, such as MAF1, to the nucleo- Conclusion
skeleton, possibly bringing them into close association with
A more detailed understanding of the animal nucleo-
specific regions of the genome. However, as yet, we do not
skeleton, NE and NPCs as well as identification of further
know the function of MAF1.
components will clearly help inform the plant field. On
the other hand there appear to be significant differences.
These differences are not only important for understand-
4.4.3 DNA Sequences of the Nucleoskeleton
ing the structural and functional organization of the plant
and Proteins That Bind Them
nucleus, but could also provide important clues to under-
standing this immensely complex organelle in all other
The identification of MARs and the discovery that some
eukaryotes.
genes that code for proteins involved in the same pathway
are clustered (Loc and Str€atling 1988), together with the
demonstration of a loop-like organization of chromatin, Acknowledgements This work was supported by grants from the
suggested that one function of the nucleoskeleton could be Biotechnology and Biological Sciences Research Council, UK, grant
numbers BB/E015735/1 and BB/G011818/1 (MWG and JF), and grants
to organize chromatin into structural domains. MARs clearly
for EK by Russian Federation for Basic Research and the Program of
exist in plants (Hall et al. 1991; Breyne et al. 1992) and there the RAS Presidium “Molecular and Cell Biology”. Thanks to Christine
is evidence that incorporation of MARs into transgenes can Richardson (Durham University) for Fig. 4.7.
60
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 The Plant Nucleolus
5
Peter Shaw

Contents 5.1 Introduction and History

5.1 Introduction and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
5.2 Functional Organization of the Nucleolus . . . . . . . . . . . . . . . 66
The nucleolus is where the cell produces ribosomes and
5.2.1 Organization of rDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 ribosomes are required by the cell in prodigious numbers.
5.2.2 rDNA Transcription and Ribosome Biogenesis . . . . . . . . . . . . 68 For example, a yeast cell contains about 200,000 ribosomes,
5.3 Assembly and Dynamics of the Nucleolus . . . . . . . . . . . . . . . 70 and has a generation time of about 100 min; thus a rapidly
dividing cell must make 2,000 ribosomes per minute. This
5.4 Epigenetics and Nucleolar Dominance . . . . . . . . . . . . . . . . . . . 70
means that 60% of the cell’s total RNA transcription is of the
5.5 Non-conventional Nucleolar Functions . . . . . . . . . . . . . . . . . . 71 ribosomal DNA (rDNA) alone. Additionally, about 80 ribo-
5.5.1 Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
5.5.2 mRNAs and Nonsense-Mediated mRNA Decay (NMD) . . 72
somal proteins must be synthesised and imported into the
5.5.3 Nucleolar Translation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 nucleolus for each ribosome made. Typically yeast nuclei
5.5.4 Other RNA Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 have about 150 nuclear pores, so each pore must import
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 about 1,000 ribosomal proteins and export about 25 ribo-
somal subunits per minute (Warner 1999). Similar
considerations apply to other eukaryotic cells, but with a
large plant cell probably requiring several million ribo-
somes. Thus the majority of an actively dividing cell’s
metabolic activity is devoted to ribosome biogenesis, and
most traffic in and out of the nucleus is targeted to or from
the nucleolus. It is therefore not surprising that the nucleolus
is the most prominent and easily observable structure within
the nucleus (Figs. 5.1 and 5.2). It has been studied for more
than 200 years, is still an active subject of research and is
still generating surprising discoveries.
The first mention of the nucleolus was by Fontana (1781),
but the name ‘nucleolus’ was coined by Valentin (1839); it
means literally little nucleus, and he described most cells as
having a nucleus within the nucleus. Heitz (1931), in a study
of plants, was the first to show the correlation between the
number of secondary constrictions in the chromosomes –
regions that appear as gaps in metaphase chromosomes and
where DNA was not detected by the Feulgen stain – and the
number of nucleoli that reappear immediately after cell
division. McClintock (1934), studying maize, then showed
that this region alone was sufficient to generate a nucleolus
P. Shaw (*)
and proposed that the chromatin at the secondary constric-
Cell and Developmental Biology Department, John Innes Centre, tion was the genetic element that organized the nucleolus,
Colney, Norwich NR4 7UH, UK now called the nucleolar organizing region or NOR. In the
e-mail: peter.shaw@jic.ac.uk

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 65

DOI 10.1007/978-3-7091-1160-4_5, # Springer-Verlag Wien 2013
66
Fig. 5.1 Different microscopical views of plant nucleoli. (a) Differen-
tial interference contrast image of isolated tobacco nuclei clearly shows
the nucleoli and nucleolar vacuoles or cavities within most nucleoli.
Bar ¼ 10 mm. (b) A single confocal optical section through pea root
tissue stained with the DNA dye DAPI shows the heterochromatin in
the nucleoplasm. The much more decondensed, highly transcribed
DNA in the nucleoli stains at a very low level with DAPI giving the
nucleoli the appearance of voids in the nuclei. Bar ¼ 5 mm. (c) Ultra-
thin section transmission electron micrograph of pea root tissue. The
nucleolus appears more electron dense than most of the nucleoplasm,
with the nucleolar vacuole or cavity staining lightly. The dense fibrillar
component and granular component show a different texture, the GC
being more open. Bar ¼ 2 mm. No nucleolus, N nucleus, NV nucleolar
vacuole, NE nuclear envelope, FC fibrillar centre, DFC dense fibrillar
component, GC granular component, CB Cajal body
early 1960s it was established by a number of groups that the
nucleolus is the site of ribosomal RNA transcription and
ribosome biosynthesis; one of the first demonstrations was
by Birnstiel et al. (1963) in pea nucleoli. Later in the decade
Miller and Beatty (1969) developed a spreading technique
for electron microscopy to produce beautiful images of the
rDNA genes (¼ Miller spreads), each gene showing up to
100 attached RNA polymerases, and the RNA transcripts
increasing in length as the polymerases progressed along the
genes (see Fig. 5.3b). The resulting images have appeared in
reviews and text books ever since, and have often been
likened to ‘Christmas trees’. The next 20 years saw a relative
decline of interest in the nucleolus as increasingly powerful
molecular biology techniques made it possible to study
single copy genes; in fact the multiple tandem repeats in
NORs still present significant problems for modern molecu-
lar biology techniques. From the mid-1990s on there has
been a resurgence of interest in the nucleolus prompted
initially by radical improvements in cell biology and imag-
ing techniques, and still more recently by coupling cell
biology to mass spectrometry-based proteomics (Andersen
P. Shaw
Fig. 5.2 Diagrammatic comparison of typical mammalian (a) and
plant (b) nucleoli. The DFC is much smaller and more densely staining
in mammalian nucleoli, whereas it generally constitutes a large propor-
tion of the volume of plant nucleoli. Transcription sites are scattered
through the DFC in both cases. Bar ¼ 1 mm. TS transcription site, DFC
dense fibrillar component, GC granular component, FC fibrillar centre
et al. 2002; Pendle et al. 2005). Live cell imaging appro-
aches, particularly using green fluorescent protein (GFP),
have shown that the nucleolus, in common with other sub-
nuclear structures, is far more dynamic than had been previ-
ously appreciated (Phair and Misteli 2000).
In this chapter we shall summarize the current state of
knowledge of the nucleolus, with particular reference to
recent developments. Where appropriate we shall concen-
trate on work from plants. In some respects there seem to be
significant differences between plant nucleoli and those of
other kingdoms, but our knowledge is still so incomplete that
it is impossible to tell whether the differences are fundamen-
tal or merely apparent. The key reviews of the nucleolus in
the ‘classical era’ are by Busch and Smetana (1970) and
Hadjiolov (1985). Recent reviews include Raska et al.
(2006a, b).
5.2 Functional Organization of the
Nucleolus
The nucleolus has a substantially different biochemical com-
position from the rest of the nucleus, giving it a different
refractive index. Nucleoli are thus easily visible by phase or
differential interference contrast microscopy. In many plant
cells the nucleolus is almost spherical in shape, and often
has a central, internal region called the nucleolar vacuole or
cavity, also visible by optical microscopy (Fig. 5.1a). Using
DNA-specific dyes such as DAPI and epifluorescence
microscopy, the nucleolus usually appears as a dark region,
suggesting it lacks DNA (Fig. 5.1b). In fact the nucleolus is
the most transcriptionally active region of the nucleus, and
therefore must contain substantial numbers of active genes.
The relatively low level of DNA labelling shows that these
active genes are highly decondensed; what is mainly seen
with DNA stains is condensed, mostly inactive DNA.
5 The Plant Nucleolus
Fig. 5.3 rDNA and rRNA organization. (a) Organization of a
single rDNA repeat. (b) Typical EM spread image, showing the
path of the gene, the increasing length of attached transcripts along
the gene and the terminal knobs. Bar ¼ 1 mm. (c) Organization of
Electron microscopy (EM) of intact nucleoli has proved
fairly uninformative about their functional organization, since
the structures seen cannot easily be interpreted in molecular
terms. This is disappointing, as spread preparations clearly
show that the structures of active transcription units are within
the resolution achievable by EM (Miller and Beatty 1969). The
problem in resolving functional units within nucleoli must lie
with the great level of compaction of the structures in vivo.
When ultra-thin sections of mammalian and various other
animal cells are stained with standard EM stains (osmium
tetroxide, uranyl acetate, lead citrate etc.) a characteristic
nucleolar substructure is often seen (Shaw and Jordan 1995).
This consists of one or more lightly staining structures, often
with a fibrous appearance, called fibrillar centres (FC),
surrounded by a layer of densely staining material called the
dense fibrillar component (DFC). The rest of the nucleolus
consists of granules about the size of ribosomes, and is termed
the granular component (GC). There has been a tendency to
interpret all nucleoli in terms of this so-called tripartite struc-
ture. However in reality there is great variety in the EM ultra-
structure between different animals and even between different
cell types and metabolic state of cells within the same species.
In plants, structures resembling fibrillar centres are often, but
not always, seen. They are embedded in a region of the nucleo-
lus which has a somewhat fibrillar texture, assumed to be the
DFC, but which does not generally stain intensely as does
mammalian DFC, and which can often only be distinguished
from the enveloping granular component by a difference in
texture (Fig. 5.1c). The DFC region of plant nucleoli is typi-
cally a much larger fraction of the total nucleolar volume than
in mammalian cells (Shaw and Jordan 1995) (Fig. 5.2).
67
the initial pre-rRNA transcript and its processing to remove the
transcribed spacers. NTS non-transcribed spacer, ETS external tran-
scribed spacer, 1 internal transcribed spacer 1, 2 internal transcribed
spacer 2
5.2.1 Organization of rDNA
Three out of the four eukaryotic ribosomal RNAs (18S, 5.8S
and 28S) are transcribed by RNA polymerase I (pol 1) from
the tandem rDNA repeats in the nucleolus. The fourth ribo-
somal RNA, 5S, is transcribed by RNA polymerase III from
tandem repeats elsewhere in the nucleus and imported into
the nucleolus (Highett et al. 1993a). Given a transcription
rate estimated to be about 40 nt/s (Kos and Tollervey 2010),
it is clear that a single rDNA copy could not provide enough
primary transcripts for the cell’s ribosome requirements.
Thus all eukaryotes have multiple copies of the rRNA
genes. In certain specialized cells, like amphibian oocytes,
extrachromosomal amplification of rDNA occurs. In virtu-
ally all eukaryotes the rDNA copies occur as tandem repeats
(Hadjiolov 1985); the reason for this is unknown, but it is
tempting to speculate that tandem repeats are more likely to
produce high local concentrations of the various factors
necessary for transcription and subsequent transcript
processing and ribosome assembly – and that this is essen-
tially what constitutes a nucleolus (Melese and Xue 1995).
In fact multiple tandem repeats, a visible nucleolus and even
pol I transcription are not strictly necessary for ribosome
biosynthesis, since a pol I deficient yeast strain in which the
rDNA is transcribed from a plasmid by pol II have been
created (Oakes et al. 1993). In these mutants the typical
crescent shaped yeast nucleolus was absent and instead a
number of bodies termed mini-nucleolar bodies were
observed. In order to form a normal nucleolar structure,
however, it seems that pol I transcription of repeated
rDNA copies is required.
68
The rDNA repeat contains a transcribed region that gives
rise to a 45S pre-rRNA transcript (35S in yeast) and an
intergenic region, often called the non-transcribed spacer
(NTS), that contains promoter and enhancer elements. In a
number of species it has been shown that a second upstream
promoter can produce low levels of a transcript that includes
the sequence of the major promoter. The pre-rRNA tran-
script contains a leader sequence, the 50 external transcribed
spacer (50 -ETS), which is removed after transcription, the
small subunit s-RNA, 18S, two internal transcribed spacers
(ITS1 and ITS2), which flank the 5.8S RNA, and finally the
large subunit l-RNA 28S followed by a short 30 external
transcribed spacer (Hadjiolov 1985) (Fig. 5.3a). The order
of the RNA transcripts and their sequences are highly
conserved, but the spacers, both transcribed and non-
transcribed, are very variable even between closely related
species. The NTS is about 2–3 kb in plants, but much
longer—20–30 kb—in vertebrates. The transcribed spacers
are also longer in vertebrates, particularly in birds, than in
plants. The primary 45S rRNA transcript is processed to
remove leader, tail and intergenic sequences in an ordered
process (Fig. 5.3c).
The number of copies of the rDNA is highly variable
throughout the eukaryotes. Mammals typically have a couple
of hundred copies, whereas most plants have several thou-
sand copies. One study estimated that only about 5% of these
copies were actively transcribed in pea root cells, and the
reason for such large numbers of repeats is unknown
(Gonzalez-Melendi et al. 2001). There is evidence that in
the human genome some rDNA repeat copies are inverted
and may not be functional (Caburet et al. 2005), but this has
not been fully confirmed as yet. Fibre fluorescence in situ
hybridization (FISH) of NORs in rice has suggested a
regular pattern of rDNA repeats, apparently without obvious
inversions or rearrangements (Mizuno et al. 2008). In fact, it
is a glaring omission that the NORs have not been fully
sequenced in any organism, due to the difficulties of
sequencing large repetitive regions with current technology,
so we have no real idea what proportion of the rDNA genes in
any plant or animal are functional or what other sequences
might be hidden in the intergenic regions.
FISH has been used extensively to examine the location of
the rDNA in well-preserved, fixed tissue. In plants this
generally shows a few dense ‘knobs’ or concentrations of
rDNA around the periphery of the nucleolus together with
some fainter labelling within the nucleolus. The knobs
correspond to the inactive rDNA copies which remain
condensed as heterochromatin, their number usually
corresponding to the number of NORs, while the active
copies are decondensed within the body of the nucleolus. In
some species, such as the diploid species rye, the internal
path of the decondensed rDNA can be clearly seen, whereas
in the closely related species hexaploid wheat, the internal
P. Shaw
labelling is more complex and may contain small condensed
regions of rDNA, while some NORs remain inactive and
unassociated with the nucleolus (Leitch et al. 1992). In pea,
the four NORs all contribute to the nucleolus, and the size of
the knobs varies inversely with the size and presumed activ-
ity of the nucleolus, showing that increased nucleolar activity
causes more of the rDNA copies to decondense and become
active (Highett et al. 1993b).
Only the NORs that were active during the previous
interphase produce secondary constrictions on mitotic chro-
mosomes, and these NORs are also stained by silver salts in
the so-called Ag-NOR labelling. In animals it has been
established that active rDNA copies are associated with
binding of upstream binding factor (UBF), a DNA binding
protein containing a number of high mobility group (HMG)
protein motifs, which are the DNA binding domains. Mais
et al. (2005) integrated arrays of ectopic UBF heterologous
binding sequences into human chromosomes and showed
that they bound UBF and some pol I components. These
pseudo-NORs gave silver-positive secondary constrictions
in the metaphase chromosomes, showing that UBF binding
is responsible for generating the decondensed rDNA seen in
the secondary constrictions and for their Ag-NOR labelling.
No UBF homolog has yet been identified in plants, but the
equivalent behaviour of plant NORs strongly indicates that
such a homolog must exist.
5.2.2 rDNA Transcription and Ribosome
Biogenesis
The location within the nucleolus of the actively transcribed
genes has been a matter of intense debate over about
25 years; see Raska et al. (2006b) for a recent summary.
Most of this debate has centred around their location with
respect to the EM ultrastructure, with some groups main-
taining that all transcription takes place in the FCs and others
that it is within the DFC. Early studies using radioactive
tritiated uridine labelling to locate incorporation into nascent
RNA in the nucleolus showed predominant labelling of
the DFC. However, Scheer and Rose (1984) showed by
immunogold labelling that the FCs contained concentrations
of RNA pol I, and that little was detected elsewhere in
the nucleolus. This was followed by various immunogold
studies that showed DNA in the FCs; see Scheer and
Weisenberger (1994) and Shaw and Jordan (1995) for
summaries. The problem with all these latter studies is that
most rDNA and most pol I is inactive at any given time. It is
only a small proportion, perhaps just a few percent, that is
active. In order to locate the active genes, nascent rRNA
must be localized. Unfortunately the tritiated uridine method
lacked both resolution and sensitivity. With the introduction
of bromo-uridine as a marker for nascent RNA, supplied to
5 The Plant Nucleolus
Fig. 5.4 Silver-enhanced 1 nm gold labelling of BrU in nascent
transcripts in pea root tissue. (a) View of an entire nucleolus showing
dense labelling of transcript sites within the DFC. Bar ¼ 1 mm.
(b) Higher magnification electron micrograph showing five
clusters of 1 nm gold particles. The clusters are approximately
the cell as BrUTP, it became possible to examine the nucle-
olar transcription sites with much greater sensitivity and
resolution (Dundr and Raska 1993; Hozak et al. 1993;
Wansink et al. 1993). This labelling showed many foci
within the DFC region of the nucleolus which sometimes
contacted the periphery of the FCs (Hozak et al. 1994).
Similar results were obtained in plants, where the more
extensive and less dense DFC made the results even more
unequivocal (Melcak et al. 1996; Thompson et al. 1997).
Double FISH labelling of the NTS and transcribed rDNA
region in peas and comparison with BrUTP transcript label-
ling showed that the transcribed DNA overlapped well with
the transcript labelling as expected, but that the intergenic
NTS labelling had very little overlap with the transcribed
region. This suggests that the transcribed genes are in
an extended conformation (Thompson et al. 1997). In a
subsequent study in pea roots, Gonzalez-Melendi et al.
(2001) showed by thin section EM that 1 nm gold labelling
of BrU consisted of discrete elongated clusters of label,
about 300 nm in length (see Fig. 5.4). The clusters were
often approximately conical in shape, and the authors
suggested these corresponded to individual transcription
units – condensed Christmas trees – compacted by a factor
of 5–8 compared to Miller spreads. Similar conclusions were
reached by Koberna et al. (2002) in animal nucleoli.
The external and internal spacers are removed from the
pre-rRNA in an ordered series of cleavage and trimming
steps, which has been well studied in yeast, but less studied
in other species. The rRNAs are also modified at numerous
sites by 20 -O-ribose methylation and pseudouridylation. The
reason for this is unclear, but the majority of the changes are
in the ribosome active site, and are thought to improve
ribosome efficiency. The site of each modification and
cleavage is specified by a cognate guide small nucleolar
RNA (snoRNA) about 60–150 nt in length, which contains
a complementary sequence to the target sequence in the
rRNA. The initial cleavage of the pre-rRNA involves U3,
U14, MRP, snR10 and snr30 and the resulting cleaved
69
conical in shape and contain 20–30 particles. Bar ¼ 100 nm. (c)
Diagram of proposed interpretation of the 1 nm gold labelling of
nascent transcripts, drawn to scale (see Gonzalez-Melendi et al.
2001). Bar ¼ 50 nm. S examples of silver-enhanced 1 nm gold
particles
products are cleaved by specific exonucleases Rat1p,
Xrn1p and the exosome (Fatica and Tollervey 2002, 2003).
The methylations and pseudouridylations are catalysed by
fibrillarin and dyskerin respectively (Nop1p and Cbf5p in
yeast). Methylations are guided by box CD snoRNAs
(containing RUGAUGA and CUGA elements) and
pseudouridylations by box H/ACA snoRNAs (containing
ANANNA and ACA elements) (Kiss 2002). An EM struc-
ture for a box CD snoRNP has been determined recently for
an archeon by Bleichert et al. (2009). Many of these
snoRNAs and cleavage intermediates have been identified
and localized in plant nucleoli (Brown et al. 2003; Kim et al.
2010). The early stages of processing, in which the 50 -ETS
was present, were found closely enveloping the transcription
foci in the DFC, whereas the later stages of processing,
where the ITS sequences were still present, were found
further away from the transcription sites, in regions broadly
corresponding to the GC (Shaw et al. 1995; Beven et al.
1996; Brown and Shaw 1998, 2008). Thus the current model
is of a vectorial distribution of processing steps with early
steps close to transcription sites and successive steps
displaced further outwards from them. Little analysis has
been carried out on the later biochemistry of ribosome sub-
unit assembly and export in plants, but the principles are
assumed to be the same as in animals and in yeast, which has
been analysed in the most detail. The RNA cleavages, in
yeast at least, are begun co-transcriptionally (Kos and
Tollervey 2010). The s-RNA is processed into the small
ribosomal subunit. The terminal knobs that are seen in Miller
spreads initially are about 15 nm in size, but become larger –
about 40 nm – and at this stage represent the small subunit
processome complexes (Dragon et al. 2002; Bernstein et al.
2004; Osheim et al. 2004). Little is known about the
corresponding processing complex for the large subunit
although a pre-60S particle has been imaged at high resolu-
tion in the EM (Nissan et al. 2004). The large and small
ribosomal subunits are exported independently to the
cytoplasm.
70
5.3 Assembly and Dynamics of the
Nucleolus
The nucleolus disassembles at the end of the G2 phase of the
cell cycle as most transcription ceases and the nuclear enve-
lope breaks down and reassembles with the onset of rDNA
transcription at the beginning of G1. The GC components
are lost from the disassembling nucleolus first, followed by
the DFC (Gautier et al. 1992). Subunits of pol I and other
DNA binding factors such as UBF remain with the rDNA
arrays; the presence of UBF alone is sufficient to produce a
secondary constriction in the mitotic chromosome (Prieto
and McStay 2008). Some nucleolar components diffuse
throughout the mitotic cytoplasm, whereas others, such as
the protein B23, associate with the periphery of the mitotic
chromosomes as chromosomal ‘passengers’ (Hernandez-
Verdun and Gautier 1994). When rDNA transcription is
halted during mitosis, unprocessed pre-rRNA transcripts
persist through the mitotic cell, demonstrating that pre-
rRNA transcript processing is also halted.
At the end of mitosis, the nucleolus reforms. First, small
round bodies, called pre-nucleolar bodies are formed
(Gimenez-Martin et al. 1974; Angelier et al. 2005;
Hernandez-Verdun 2006). When transcription of the rDNA
is reinitiated, the pre-nucleolar bodies (PNBs) disappear as
new nucleoli are formed. Originally it was assumed that the
PNBs condensed onto the active rDNA, but recent data
obtained using the optical technique FRAP (fluorescence
recovery after photobleaching) suggest rather that pre-
rRNA processing complexes (and also unprocessed pre-
rRNA) are preassembled in the PNB, and then diffuse out
of the PNBs, associating with the reforming nucleoli. Where
more than one active NOR is present in the nucleus, separate
nucleoli generally initially form at each active NOR in early
G1. These small nucleoli then have a tendency, especially in
plants, to fuse together to a single nucleolus as interphase
progresses (Shaw and Jordan 1995).
Recent dynamic studies using a variety of nuclear and
nucleolar proteins marked by GFP have led to a complete
reassessment of the interpretation of nucleolar and nuclear
structure. For many years cell biologists have visualized
fixed cells and have thus had a tendency to regard the
structures seen as stationary and long-lived. However live
cell imaging studies have revealed a much more dynamic
picture. First, sub-nuclear structures themselves move and
rearrange themselves within the nucleus, and the nucleus
itself moves and changes shape. For example, Boudonck
et al. (1999) showed in Arabidopsis that Cajal bodies move
and fuse together, changing their positions and number. At
the molecular level, all nucleolar and nuclear proteins are
in constant flux, exchanging between the nucleolus and
cytoplasm, and the mean nucleolar residence time of even
P. Shaw
well-characterised ‘nucleolar’ proteins is only a few tens of
seconds (Phair and Misteli 2000; Misteli 2001; Olson and
Dundr 2005). The distinction between ‘nuclear’ and ‘nucle-
olar’ proteins lies in their residence time in the nucleolus,
with nucleolar proteins spending a greater proportion of their
time in the nucleolus. The structure and even the existence of
the nucleolus as a discrete structure must depend on the
rDNA nucleating a small sub-population of proteins that
then form a structure on which all the other proteins assem-
ble and disassemble dynamically. The nucleolus (and other
nuclear bodies such as Cajal bodies) thus represent a steady
state flux of proteins in rapid equilibrium with the surr-
ounding nucleoplasm (Raska et al. 2006a). It is even possible
that the DNA in the nucleolus is in dynamic equilibrium with
the rDNA at the nucleolar periphery; this has yet to be tested
in living cells.
5.4 Epigenetics and Nucleolar Dominance
Not all rRNA genes are necessarily transcriptionally active
in a given nucleolus. In fact in most plants, the vast majority
are not transcribed. We assume that all rDNA copies are
identical and potentially transcribable (but as mentioned
above, this is by no means certain). Current evidence, how-
ever, is that rRNA genes may be in one of three states:
(1) inactive and condensed into heterochromatin, which in
plants is mostly seen as knobs of heterochromatin at the
nucleolar periphery, but also within the nucleolus; (2) active
and transcribed, in an extended conformation within the
nucleolus; finally (3) an ‘open’, poised conformation –
potentiated and available for transcription, but not currently
transcribed (Huang et al. 2006; McKeown and Shaw 2009).
The balance between these states, and ultimately the level of
pol I loading, may depend on DNA methylation, differences
in the histone variants associated with the DNA, remodelling
of the DNA, particularly the promoter regions, and the
presence of histone modifications (Grummt and Pikaard
2003). rDNA can retain the level of pol I association through
mitosis, and so the chromatin state of rDNA can be epige-
netically inherited.
Nucleolar dominance is a particularly striking effect that
is seen in hybrid organisms, where it is often found that the
NORs of one parental genome are silent while those of the
other are active (see Tucker et al. (2010) for a recent review).
This is usually ascribed to the suppression of the under-
dominant genome by the dominant one, but recent evidence
from a wheat-rye hybrid has suggested that the NORs from
the dominant genome are also up-regulated (Silva et al.
2008). Nucleolar dominance has been observed in many
plants and animals, but has been most thoroughly studied
in plants, probably because inter-species hybrids are much
easier to study in plants than animals. In a given hybrid
5 The Plant Nucleolus
cross, the same member of the pair is always under-
dominant or dominant, irrespective of which parent provides
which genome. This means that nucleolar dominance is not
mediated by an equivalent mechanism to parental imprint-
ing, nor to X chromosome inactivation, in which a random
choice of inactive chromosome is made. In Brassica hybrids,
it has been demonstrated that nucleolar dominance is
disrupted by aza-deoxycytidine, which reduces DNA meth-
ylation, and by Trichostatin A, a histone deacetylase inhibi-
tor which leads to an increase in histone acetylation.
Significant progress in understanding nucleolar domi-
nance has been made using Arabidopsis suecica, a hybrid
of A. thaliana and A. arenosa. In young seedlings of
A. suecica, the rRNA genes from both genomes are highly
expressed, but as the plant grows, the A. thaliana-derived
rRNA genes become silenced. This suggests that nucleolar
dominance may be an aspect of active gene dosage control
mechanisms, where different levels of rRNA are required at
different stages of development (Tucker et al. 2010). RNAi
has been used systematically to determine which histone
modifying enzymes are required for gene silencing in nucle-
olar dominance. This approach has pinpointed the histone
deacetylases HDT1 and HDA6, the de novo DNA met-
hyltransferase DRM2, and the methylcytosine binding
domain proteins MBD6 and MBD10 (Preuss et al. 2008).
MBD6 is presumed, by analogy with animal studies, to
participate in the formation of heterochromatin, but this
has not yet been formally shown in plants. DRM2 is part of
the RNA-directed DNA methylation pathway, in which
double stranded templates are formed from pol IV RNA
transcripts by the RNA-dependent RNA polymerase,
RDR2, diced into 24 nt siRNAs, which then guide DRM2
to methylate the homologous DNA sequences. In confirma-
tion that this pathway is indeed involved in nucleolar domi-
nance in plants, knockdown of RDR2, DCL3 as well as
DRM2 disrupted the rRNA silencing of the A. thaliana
derived rRNA genes in A. suecica (Preuss et al. 2008).
There is also evidence for RNA-mediated silencing of
rRNA genes in mammals, where rRNA genes are silenced
by the nucleolar remodelling complex, NoRC, which is
recruited to a subset of rRNA genes by 200–300 nt RNA
species, termed pRNA, which themselves derive from
intergenic regions of rDNA (Mayer et al. 2008; Santoro
et al. 2010). Thus although the detailed mechanisms may
differ, in both plants and animals control of rRNA gene
expression depends on RNA-mediated silencing by sequ-
ences derived from the intergenic rDNA, which is presum-
ably expressed from the minor intergenic rDNA promoter.
As yet, however, little is known about how the subset of
genes to be silenced is chosen, and why one particular
genome is dominant or under-dominant. In hybrids, it is
tempting to speculate that this may be due to the relative
strength of interactions between the silencing RNAs and the
rDNA of the two genomes.
71
5.5 Non-conventional Nucleolar Functions
5.5.1 Proteomics
High throughput proteomics has now been applied to
purified nucleoli in a number of studies including humans
(Andersen et al. 2002, 2005; Scherl et al. 2002) and the plant
A. thaliana (Pendle et al. 2005). These studies have uncov-
ered an enormous range of several hundred proteins as
nucleolar constituents, and has added a new dimension to
the previous observations indicating that the nucleolus is the
site of many other functions than ribosome biogenesis
(Pederson 1998). In this type of proteomic analysis of com-
plex mixtures, the question of possible contaminants imme-
diately arises. Pendle et al. (2005) answered this question by
localizing GFP fusions in vivo of a substantial, randomly
chosen set of proteins identified in the nucleolar fractions
(see Fig. 5.5). The vast majority (87%) were indeed located
in the nucleolus, but most were also seen in other parts of the
nucleus or cytoplasm, as would be expected. In fact since the
dynamic studies mentioned above have shown that virtually
all nuclear proteins at least visit the nucleolus, there is a real
question of what actually constitutes a nucleolar protein. The
best approach is to compare the nucleolar protein profile
quantitatively with the nuclear and cytoplasmic protein
profiles, and thus arrive at a nucleolar partition ratio for
each protein. This has been done for human cell culture
nucleoli using stable isotopic labelling of the different
fractions – SILAC – prior to mass spectrometry analysis
(Boisvert et al. 2009). Given the rapid diffusion of most
proteins in and out of the nucleolus, it is fair to ask how
nucleoli can be purified at all. The answer to this is not clear,
but it is presumably because the breakage of the cell and
nuclear membranes that precedes nucleolar purification must
also alter the solution conditions to prevent most proteins
from diffusing away from the nucleoli. However the caveat
that proteins may be selectively lost during nucleolar isola-
tion is important, and shows the need to complement prote-
omics approaches by in vivo studies of specific proteins.
SILAC methods have also been used to analyse the
dynamics of nucleolar proteins after treatment with specific
drugs or stresses (Lamond and Sleeman 2003; Andersen et al.
2005), during the cell cycle (Leung and Lamond 2003), and
after viral infection (Emmott et al. 2010; Hiscox et al. 2010).
As an example, proteomic analysis of nucleoli after treatment
with various inhibitors of the proteasome showed a large
accumulation of ribosomal proteins. Photobleaching
experiments of individual GFP-tagged ribosomal proteins
showed that these proteins are synthesized and imported to
the nucleolus very rapidly. The number of rRNA molecules
needs to be balanced with the number of ribosomal proteins
since they are required in stoichiometric amounts (Rudra and
Warner 2004). These experiments suggest that this balance is
72
Fig. 5.5 Examples of expression of GFP-fusion constructs for proteins
found in proteomic analyses of Arabidopsis nucleoli. Transient expres-
sion in Arabidopsis culture cells (see Pendle et al. 2005). (a) EJC
component Y14. (b) EJC/export factor ALY/REF. (c) Splicing factor
PRP19 shows a perinucleolar distribution. (d) Protein of unknown
function localized to nucleolar substructures. (e) Protein of unknown
function localized to nucleolus and other nuclear bodies. Bar ¼ 5 mm
achieved by making an excess of the r-proteins and
ubiquitinating and degrading any that remain unincorporated
into ribosomes (Andersen et al. 2005).
5.5.2 mRNAs and Nonsense-Mediated mRNA
Decay (NMD)
A detailed analysis of the nucleolar proteome from Arabi-
dopsis showed that many proteins involved in mRNA splicing
and translation were present in the nucleolus. In particular, it
was striking that almost all the known components of the post-
splicing exon-junction complex (EJC) were detected, and
were subsequently shown by GFP fusions to indeed be
associated with the nucleolus (Pendle et al. 2005). In contrast,
although EJC components were detected in the human nucle-
olar proteome, localization studies have not so far confirmed
their localization (Custodio et al. 2004), suggesting possible
differences between plants and animals. The EJC is a multi-
protein complex that is deposited 20–30 nucleotides upstream
of splice junctions in mRNAs. The complex contains residual
spliceosomal proteins, as well as factors involved in mRNA
export and translation. The complex remains in place until the
pioneer round of translation (Lejeune et al. 2004). The EJC
also mediates nonsense-mediate mRNA decay (NMD)
(Lejeune et al. 2004). In the most studied mechanism, if the
ribosome during the initial round of translation encounters a
stop codon upstream of an EJC complex, the stop codon is
identified as a premature stop codon. The mRNA is thus
marked as aberrant, the NMD factors upf1, upf2 and upf3 are
recruited to the EJC complex and the mRNA is degraded,
P. Shaw
Fig. 5.6 Relative amounts of single exon, aberrantly spliced and fully
spliced mRNAs in polyA þ libraries made from whole cells, nuclear
extracts and nucleolar extracts respectively. Whereas the single exon
mRNAs are present in about the same proportion in each library
(15–20%), the percentage of aberrantly spliced mRNAs increases
from a very low level in whole cells, mainly from cytoplasmic
mRNA (2%), to an intermediate level in nuclear extracts (13%), and
the highest level in nucleolar extracts (38%). Thus the aberrant mRNAs
purify predominantly with the nucleolar fraction (see Kim et al. 2009)
probably mostly through cytoplasmic P bodies (Parker and
Sheth 2007; Xu and Chua 2009).
The observation of EJC components in the nucleolus in
plants suggests that mRNAs may also be located there. This
has been confirmed by constructing cDNA libraries from
polyA + RNA extracted from purified nucleoli (Kim et al.
2009). Many individual clones were sequenced from the
nucleolar library and compared with similar libraries made
from purified nuclei and entire cell extracts respectively.
Remarkably, this analysis showed that mis-spliced and oth-
erwise aberrant mRNAs were greatly enriched in the nucle-
olar extract – about ten-fold compared to the entire cell
extract, which would be expected to be mainly cytoplasmic
(see Fig. 5.6). Single exon transcripts, which do not undergo
splicing, were found in the same amounts in all three
libraries, showing that contamination could not explain
these results. The aberrant transcripts contained all sorts of
splicing errors, including intron retention and splice bound-
ary mis-sensing of all types. It might be argued that many of
these species were alternatively spliced rather than mis-
spliced. Since alternative splicing has been little studied in
plants, this is difficult to assess. However, the spliced
variants were at odds with the standard gene models from
the genome sequence, most of which have been verified by
EST and other mRNA sequences. Thus it is likely that most
of these species should be considered as mis-spliced rather
than alternatively spliced. About 90% of the aberrant
transcripts fulfilled the conditions for targeting for NMD,
at least according to the mammalian NMD criteria. Finally
Kim et al. (2009) showed by GFP fusion analysis that the
NMD factors upf3 and upf2 were localized to the nucleolus,
although upf1 was not. These results strongly argue that the
nucleolus is involved in mRNA surveillance and export.
5 The Plant Nucleolus
In fact the nucleolus has been previously implicated in
mRNA export in experiments going back to the 1960s
(Pederson 1998). Harris (1967) showed that in hetero-
karyons between chicken erythrocytes and human HeLa
cells that no proteins of chicken origin were produced until
the previously inactive chicken nucleus reformed a nucleo-
lus, suggesting that the lack of a functional nucleolus
impaired mRNA export from the chicken nucleus. More
recently, the nucleolus in transport-defective yeast mutants
has been shown to be disrupted (Schneiter et al. 1995), and
heat shock or mutation of nucleolar proteins lead to accu-
mulation of polyA + RNA in the nucleolus (Kadowaki et al.
1995). Ideue et al. (2004) have shown that a subset of poly
A + mRNA associated transiently with the nucleolus during
export, and an intron-containing transcript accumulated
in the nucleolus in export-deficient mutants, whereas tran-
scripts from the intronless cDNA did not.
5.5.3 Nucleolar Translation?
The finding that mis-spliced mRNAs are preferentially
concentrated in the nucleolus, whether they are degraded
there or simply pass through on their way to the cytoplasm,
raises the interesting question of how these RNA species are
identified. The best studied mechanism for NMD requires a
ribosome to detect a premature termination codon during the
initial pioneer round of translation. If such transcripts are
identified before nuclear export and sent to or preferentially
retained in the nucleolus as the current data imply, this
mechanism would require at least the pioneer round of trans-
lation for some mRNAs to take place in the nucleus or
nucleolus or both. This is a controversial idea, but one that,
surprisingly, has some experimental support. There is evi-
dence for the presence of amino-acylated tRNAs in the nucle-
olus of yeast (Steiner-Mosonyi and Mangroo 2004), and in the
nucleus of Xenopus oocytes (Lund and Dahlberg 1998). The
nucleolus is also full of ribosomes, some of which could be
competent for translation. Evidence for actual protein transla-
tion in pea nucleoli was first published during the 1960s
(Birnstiel et al. 1961; Birnstiel and Hyde 1963). This work
was subsequently assumed to be due to cytoplasmic contami-
nation after it was shown that translation occurred in the
cytoplasm. However the idea was revisited using modern
cell biological methods by Iborra et al. (2001), who allowed
cells to incorporate labelled amino-acyl tRNA and then
detected the incorporated labelled amino acid residues by
fluorescence microscopy. This showed 9–15% of the labelling
within the nucleus, with prominent nucleolar labelling.
Nathanson et al. (2003) questioned these experiments,
showing that in their hands only about 1% of the labelling
was intranuclear, and suggested that the results of Iborra et al.
(2001) were due to cytoplasmic contamination. This contro-
versy has yet to be satisfactorily resolved (Iborra et al. 2004).
73
5.5.4 Other RNA Species
There is emerging evidence for a number of other non-
conventional roles for the nucleolus (Table 5.1) (Pederson
1998; Olson et al. 2002; Raska et al. 2006a). For example,
tRNA genes have been shown to be preferentially located in
or at the periphery of the nucleolus, and this location is
dependent on their transcription (Thompson et al. 2003).
The resulting transcripts are processed by trimming at 50
and 30 ends by RNAse P, an RNA-containing enzyme,
which is also found in the nucleolus (and Cajal bodies)
(Jarrous et al. 1999). Similarly Highett et al. (1993a) showed
a preferential location of 5S genes at the nucleolar periphery
by FISH. Telomerase (both RNA and protein components)
has also been found in the nucleolus, either as part of its
biosynthesis or sequestered there as a control mechanism
(Wong et al. 2002). Many RNAs, including snoRNAs,
tRNAs and telomerase RNA are modified by pseudo-
uridylation, which is catalyzed by cbf5p/dyskerin, which is
found in the nucleolus in plants, animals and yeast, and
by 20 -O-ribose methylation, catalysed by fibrillarin, also
found in the nucleolus (and Cajal bodies). Yet another
RNA complex which has been associated with the nucleolus
is the signal recognition particle (SRP). This is an RNA-
containing complex that targets the translation of certain
proteins to the endoplasmic reticulum (ER) by first blocking
and then releasing translation on binding to the SRP receptor
in the ER. Stages in the assembly of the SRP have been
shown to occur in the nucleolus by in situ hybridization,
biochemical fractionation and live cell microinjection stud-
ies (Chen et al. 1998; Jacobson and Pederson 1998; Politz
et al. 2002). Finally, the various components required for
both rDNA silencing (see above) and heterochromatic
silencing co-localize with the siRNAs themselves in the
nucleoli and in Cajal bodies (Li et al. 2006; Pontes et al.
2006). Thus the nucleolus (and Cajal bodies) are involved in
siRNA production and assembly of silencing complexes
both for rDNA and for other genes.
Why should all these other activities and complexes be
associated with the nucleolus? One clue is that they all have
the post-transcriptional processing of RNA species and asso-
ciation of the resulting RNAs with multiple proteins in
common with ribosome biosynthesis. In all cases, the RNA
protein assembly pathways also probably require a complex
series of steps involving various chaperones and accessory
factors. Clearly in some way concentrating all the factors
and processes needed to make ribosomes together in a
specialized region of the nucleus—the nucleolus—has
important benefits, and the same may apply to the biosyn-
thesis of many other multi-component RNA complexes.
In addition, some of the activities and factors necessary to
make the various RNA machines are shared between many
different processes, such as pseudo-uridylation, RNA trim-
ming, ribose 20 -O-methylation. Concentrating these factors
74
Table 5.1 Non-conventional functions of the nucleolus in RNA
metabolism and other cell processes
Partial assembly of telomerase RNP
Partial assembly of Signal Recognition Particle
50 and 30 processing of some pre-tRNAs by RNAse P
Processing and assembly of RNAse P
Processing of polycistronic pre-snoRNAs in plants
Nucleotide modifications in snoRNAs and snRNAs
Production of heterochromatin siRNAs in plants
Nucleolar phase for some mRNAs
Concentration of aberrant mRNAs/ NMD (plants)
Nucleolar trafficking of some animal and plant virus proteins
Sensor of cell stress
Sequestration of various factors – cdc14, p53/MDM2/ARF, telomerase
in the nucleolus for ribosome biosynthesis may have the side
effect of locating other processes that require some of the
same factors in the nucleolus as well.
Conclusion
Over the past 15–20 years the importance of the nucleolus
has grown with increasing understanding of the range of
activities located in this nuclear region. It is clear that it is
the major centre in the nucleus for RNA transcription and
processing and for the assembly of a wide variety of RNP
complexes. It is involved with the products of all the
DNA-dependent RNA polymerases in one way or ano-
ther, and recent evidence from plants shows that it is
likely to be involved in mRNA export and surveillance,
as well as RNAi silencing mechanisms. The extent of this
involvement and the detailed mechanisms underlying it
are the subject of active research.
The activity of the nucleolus underpins most of the
activity of the cell, and it is therefore not unexpected that
responses to growth conditions and to stresses involve
modulation and responses in the nucleolus. In human
pathology, nucleolar morphology has been used in
tumour diagnosis and grading for prognosis over many
years and an enigmatic body called the perinucleolar
compartment has emerged as closely linked to malignant
transformation (Kopp and Huang 2005). One of the major
mediators of cellular stress responses and genome dam-
age in mammalian cells is the p53 transcription factor;
more than 50% of human cancers have impaired p53
pathways, which has made its regulation the subject of
intense study. Rubbi and Milner (2003) have shown that
disruption of the integrity of the nucleolus, by targeted
UV irradiation, drug treatment or specific antibodies, can
induce activation of the p53 pathway, suggesting that the
nucleolus itself is the upstream stress sensor for DNA
damage and other stresses. It is not known whether simi-
lar mechanisms operate in plants, but recent evidence
has implicated relocation of the RNA binding factor
P. Shaw
and EJC component eiF4a-III to the nucleolus and
nuclear granules in response to hypoxia and other stresses
(Koroleva et al. 2009a, b), suggesting that the nucleolus is
indeed involved in stress responses in plants.
Research on the nucleolus has led the way in a number
of areas of cell and molecular biology. Its fundamental
importance to all eukaryotic cells means that it can pro-
vide paradigms for many activities and mechanisms. It is
also involved in biosynthesis of some of the most ancient
cellular machinery involving RNA. In the light of this,
the evolution of the nucleolus in the first eukaryotes and
its relation to the organization of equivalent processes
in archaea, from which the informational processes in
eukaryotes are thought to have developed, is likely to be
a fruitful field for future study.
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 Cell Cycle Modules in Plants for Entry
into Proliferation and for Mitosis 6
Zoltán Magyar, Masaki Ito, Pavla Binarová, Binish Mohamed,
and Laszlo Bogre

Contents 6.1 Choreography of the Cell Division Cycle

6.1 Choreography of the Cell Division Cycle . . . . . . . . . . . . . . . . 77
6.2 The Core Cell Cycle Regulators, Can One Do the Job
The cell cycle is an orderly progression through a series of
for All? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 events culminating in the duplication of chromosomes during
S-phase and in the segregation of chromosomes into daughter
6.3 The Entry Module into the Cell Cycle . . . . . . . . . . . . . . . . . . . 83
cells during mitosis. The S-and M-phases are the so called
6.4 Transcriptional Regulation at G2 Phase to Mitosis and active phases, and each is preceded by a preparatory gap
the Onset of Endoreplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
6.4.1 Genes Expressed During Late G2 and Mitosis . . . . . . . . . . . . . 87 phase, (known as G1 and G2 respectively) when regulatory
6.4.2 Transcriptional Activation of G2/M-Specific Genes . . . . . . . 87 inputs are pereceived; (Fig. 6.1). Chromosomes during G1-,
6.4.3 Transcriptional Repression of G2/M-Specific Genes . . . . . . 88 S and G2-phases (interphase) are decondensed but these
6.4.4 Other Factors Affecting the G2/M-Specific Transcription 89 intephase chromosomes are still known to occupy distinct
6.5 Spatial Organization of Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . 89 territories within the nucleus and this organization is impor-
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 tant for replication, transcription, repair and recombination
processes (Schubert and Shaw 2011). Mitosis is a cell cycle
period packed with morphological events subdivided into
pro-, meta-, ana- and telophase followed by cytokinesis.
Within this chapter we will focus on the central regulators
of the cell cycle phase transitions, but before we get onto the
regulators in detail we first summarise what is being
regulated during these cell cycle phases with an aim to
pinpoint conserved and plant-specific mechanisms.
DNA synthesis is largely conserved in eukaryotes. It
involves (i) assembly of the prereplicative complex at multiple
replication origins during G1 (ii) origin activation by firing of
these prereplicative complexes and (iii) formation of the
preinitiation complex that leads to DNA synthesis. The re-
replication of DNA during G2 is blocked by inhibiting the re-
assembly of the prereplicative complexes. This is done by the
so called licensing factor, which links to a cell cycle stage with
high activity of the central regulator, cyclin-dependent kinase
(CDK, see later). These prereplicative complexes can there-
fore only form after mitosis in G1 when CDK activity is low,
thus ensuring that the synthesis and segregation of sister chro-
matids alternate. Both the components of the prereplicative
and preinitiation complexes and the regulatory protein kinases
L. Bogre (*)
(CDK and Cdc7) are conserved in yeasts, animals and plants
Centre for Systems and Synthetic Biology, Royal Holloway, (Costas et al. 2011a). Genome wide mapping studies have
University of London, Egham Hill, Egham TW20 0EX, UK identified plant replication origins and chromatin marks which
e-mail: l.bogre@rhul.ac.uk

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 77

DOI 10.1007/978-3-7091-1160-4_6, # Springer-Verlag Wien 2013
78
Fig. 6.1 Regulatory components of the three main cell cycle
transitions, G1/S, G2/M and M/G1. At the centre of the conserved
cell cycle regulation is cyclin-dependent kinase (CDK) in complex
with the phase-specific cyclins, and opposed by CDK inhibitors
(CKI) and further regulated by positive (P-161) and inhibitory (P-
T14; P-Tyr15) phosphorylations. At the G1/S transition, D-type cyclins
(CYDs) preferentially interact with CDKA;1, and these complexes win
against the opposing CDK inhibitors (KRPs). The main target of CYCD-
CDKA;1 complex is RBR1, which is inactivated through CDKA;1
phosphorylation leading to the release of E2F transcription factors (pri-
marily E2FB) to activate genes for G1/S transition. CYCA3;1 cyclin has
also been shown to interact with CDKA;1, phosphorylate RBR1 and be
involved in the G1/S transition. G2/M is preferentially regulated by B-
type CDKs (CDKBs) in complex with A- and B-type cyclins (CYCA and
act as important regulatory elements (Costas et al. 2011b, c).
Incomplete DNA synthesis and DNA damage is sensed and
signalled to the cell cycle through the DNA damage checkpoint
and this halts cell cycle progression to allow time to repair the
DNA (Cools and De Veylder 2009; Cross et al. 2011). Some but
not all the components of the DNA integrity checkpoint are
conserved among eukaryotic organisms.
Newly synthesised sister chromatids are held together by
the cohesin ring complex. This is resolved by the release of
active separase which cleaves cohesins at the meta- to ana-
phase transition of mitosis, leading to perfect timing of sister
chromatid separation. During meiosis I sister chromatids are
held together by meiosis-specific cohesins. Cohesins and
associated proteins also help to establish the bi-orientation
of kinetochores during mitosis, while the meiosis-specific
cohesion at kinetochores establishes the mono-orientation of
sister chromatids. The molecular players and mechanisms of
sister chromatid separation during mitosis and meiosis are
largely conserved in eukaryotes, including plants (Yuan
et al. 2011). Following the onset of mitosis, chromatin
gradually becomes condensed from prophase to metaphase
driven by histone modifications, primarily histone
Z. Magyar et al.
CYCB). These mitotic CDKs are opposed by the plant-specific CDK
inhibitor, SIM. The role of inhibitory phosphorylation on CDKB is not
well understood. Interestingly, CYCD4;1 can associate with mitotic
CDKB1;1 and has been shown to trigger mitosis, possibly through
RBR1 phosphorylation. Exit from mitosis at the meta- to anaphase
transition is trigerred by the degradation of CYCA and CYCB through
the activation of the anaphase promoting complex (APC) by CDC20
during the M/G1 transition and later in G1. CYCA and CYCB levels are
kept low by the activation of the APC by CCS52 proteins. In plants, cells
can exit from proliferation, enter into a G0 state and differentiate both at
G1/S and G2/M transitions. Both these transitions are regulated by
external signals such as light, nutrient availability, hormones and devel-
opmental cues. Arrows indicate activation, hammers repression, pointing
fingers show regulatory inputs to the cell cycle
phosphorylation and deacetylation as well as molecular
motors called condensins (Costas et al. 2011b).
The segregation of chromosomes into daughter cells is
driven by the mitotic spindle. In higher plants microtubules
are nucleated at dispersed sites on existing microtubules,
membranes or chromatin (see later). In contrast to animals,
there are no microtubule organizing centres (MTOC) such
as centrosomes to control the nucleation and organization of
spindle microtubules. Instead, chromatin-mediated micro-
tubule nucleation and organization is the major mechanism
to build mitotic spindles (Karsenti and Vernos 2001). The
mitotic checkpoint monitors the full alignment of
chromosomes at the metaphase plate through the tension
exerted by the kinetochore microtubules on the sister
chromatids. This signals to the anaphase promoting com-
plex (APC) and triggers the dissolution of cohesion
molecules through the proteolysis of securin, an inhibitor
of separase. Dissolution of cohesion between sister
chromatids allows the chromosomes to separate by the
pulling force of the anaphase spindle (Morgan 2007). The
regulatory mechanisms for the mitotic checkpoint are also
conserved in eukaryotic organisms (Caillaud et al. 2009).
6 Cell Cycle Modules in Plants for Entry into Proliferation and for Mitosis
Plant cells are encased in a cell wall, and therefore build-
ing the septum inbetween two daughter cells during cytoki-
nesis is an inside out process through the expansion of a disk
like structure, called the phragmoplast. Microtubules pro-
vide the central skeleton for this structure as well as the
highway for the delivery of vesicles to the cell plate (Lloyd
2011). The timing when phragmoplast forms and its spatial
organization is controlled by cytokinetic MAPK signalling
pathways (Bogre 2011).
6.2 The Core Cell Cycle Regulators, Can One
Do the Job for All?
The cell cycle is an orderly progression through a series of
events leading to the duplication of chromosomes during S-
phase and in the segregation of chromosomes into daughter
cells during mitosis. There are three main control points on
this road; (1) the entry into S-phase, (2) the entry into mitosis
and (3) the exit from mitosis back into G1 phase (Fig. 6.1). In
simple eukaryotes, such as yeasts, there is a single control-
ler; the cyclin-dependent kinase (CDK) that governs all three
transitions. A central question is; how a single regulator can
perform such diverse tasks. In fact in more complex animals
and higher plants there has been an expansion of the CDK
and cyclin families. For example, in Arabidopsis, there are
12 members of the CDK gene family falling into 8 groups,
CDKA-F (Vandepoele et al. 2002). This grouping appears
conserved as it has also been found in other plants, such as
rice (Guo et al. 2007), lower plants including the lycophyte
Selaginella (Banks et al. 2011) and in algae (Bisova et al.
2005). Not all of these CDKs are directly involved in cell
cycle regulation; the two main groups of CDKs with cell
cycle functions are CDKA, of which there is only a single
gene in all flowering plants analysed, and CDKBs with
several members (De Veylder et al. 2007). CDKA can func-
tionally complement the yeast cdc2 mutant, and is thought to
control multiple cell cycle transitions throughout the cell
cycle (Hirt et al. 1991). However, evidence is accumulating
that in plants CDKA is predominantly specialized towards
a singly target, RBR1 for transcriptional control of cell cycle
transitions (Van Leene et al. 2011). The plant-specific
CDKB1 and CDKB2 groups are expressed from late
S-phase through G2 to M-phases and have functions during
these phases. To what degree plant CDKA and CDKBs are
specialized or carry out redundant functions is an interesting
but not fully resolved question. Pollen mitosis was found to
be delayed or absent in heterozygous cdka;1 mutants,
resulting in mature pollen with only a single sperm cell
(Iwakawa et al. 2006; Nowack et al. 2006). However,
when plants were rescued through this stage, the cdka;1
mutant was surprisingly viable, indicating that CDKA;1 is
not absolutely required for cell cycle progression.
79
Nevertheless, with only a single sperm, the double fertiliza-
tion fails because only the female egg cell is fertilized, and
not the central cell. The result is seed abortion due to the lack
of endosperm (Nowack et al. 2007; Aw et al. 2010). CDKB
is a larger gene family (Table 6.1) than A, and therefore it is
not unexpected that a cdkb2 mutant in Arabidopsis is not
fully compromised in cell division, although tissue organi-
zation and stem cell maintenance in meristems are affected
(Andersen et al. 2008). The level of CDKA activity is also an
important determinant for meristem organization in
Arabidopsis (Gaamouche et al. 2010) and reprogramming
of cell differentiation in the moss Physcomitrella (Ishikawa
et al. 2011).
It is assumed that cell cycle phase specificity is achieved
by the interchange of the cyclin regulatory subunits of
CDKs, and correspondingly, in all organisms there are mul-
tiple cyclins with cell cycle phase-specific expression
patterns and roles (Hochegger et al. 2008). In higher plants,
there has been a large expansion of the cyclin family with
around 30 members in Arabidopsis (Vandepoele et al. 2002;
Menges et al. 2007). These can largely be matched to the
animal A-, B-, and D-type cyclins. In agreement with the
phase-specific role of cyclin subunits, the expression of
cyclins is also tightly regulated; either linked to cell cycle
phases, or coupled to external signals, hormones and devel-
opmental programs (Menges et al. 2003). The sequence
similarity-based classification of cyclins can be matched
across the higher plant species Arabidopsis, rice and maize
(Vandepoele et al. 2002; Guo et al. 2007; Hu et al. 2010;
Buendia-Monreal et al. 2011), as well as to lower plants and
algae (Bisova et al. 2005; Banks et al. 2011). The transcrip-
tional regulation of cell cycle genes is also similar across
species such as Arabidopsis and rice (Menges et al. 2003;
Guo et al. 2007). How these core cell cycle genes have
evolved in the plant lineage is becoming apparent by com-
parative analysis of genome sequences from algae, moss and
Selaginella (Bisova et al. 2005; Huysman et al. 2010; Banks
et al. 2011). Moreover, having the genome sequence of
eukaryotic organisms such as Naegleria, a free living
amoeba, that is considered to be close to the base of the
tree separating plants and fungi-animal kingdoms, allows
the evolution of the conserved regulatory mechanism of the
cell cycle to be unravelled (Cross et al. 2011). This approach
holds great promise, suggesting that knowledge gained from
studying model organisms such as yeasts, human cells or
Arabidopsis can be used to infer cell cycle regulation in
other species or crops.
Is such complexity in CDK and cyclin numbers an essen-
tial feature of the core cell cycle machinery for regulating
cell cycle transitions? This was recently tested in fission
yeast, where the cell cycle control system is somewhat
simpler with a single CDK, CDC2, and a couple of G1 and
G2 cyclins. The Nurse group first replaced CDC2, and the
80 Z. Magyar et al.

Table 6.1 List of core cell cycle genes in representative sequenced plant genomes
Core cell cycle genes Description Chlamydomonas Physcomitrella Selaginella Oryza sativa Arabidopsis
CDKA Canonical cyclin dependent kinase (CDK) 1 2 1 3 1
CDKA;1 in plants complementing the yeast
Saccharomyces pombe cdc2 mutant.
Primarily interacts with D-type cyclins and
KRPs and therefore one of the main
functions is to inactivate RBR
CDKB Plant-specific CDK functions in G2- and M- 1 7 2 2 4
CDKB1 phases, the two major subclasses in
CDKB2 flowering plants are expressed during S/G2
(B1) or G2/M (B2)
CYCD Closely related to animal D-type cyclins, 3 2 3 12 10
CYCD1-7 can complement yeast G1 cyclins. There are
seven subclasses in flowering plants. Their
expression can be cell cycle phase specific
(both G1/S and G2/M) or coupled to
external stimuli, such as phytohormones,
sucrose, or to developmental cues
CYCA Related to animal A-type cyclins, there are 1 8 3 7 10
CYCA1 three main subclasses in flowering plants;
CYCA2 CYCA3s are upregulated at the G1/S
transition and can interact with CDKA;1.
CYCA3
CYCA1 and CYCA2 members are expressed
from G2- to M-phase, associate with CDKBs,
and negatively regulate endocycle
CYCB Related to animal B-type cyclins, divided 1 2 1 5 11
CYCB1 into three subclasses: B1, B2 and B3;
CYCB2 transcriptionally controlled by M-specific
activator promoter elements (called MSA)
CYCB3
and are destroyed by APC ubiquitin ligase
KRP KIP Related Proteins (KRP) are – 1 3 6 7
KRP1-7 functionally related to yeast and animal
CDK inhibitors, such as the p27KIP. Interact
with CDKA;1 and D-type cyclins, KRP1/
2 are phosphorylated by CDKA;1 and
CDKB1;1 preceding their destruction
SIM/ Plant-specific CDK inhibitors, comprising ? ? ? ? 13
SMR SIAMESE (SIM) and SIAMESE-related
(SMR) proteins. Appear to specifically
interact with and oppose the activities of
G2- and M-phase specific CDKB/CYCA or
CDKB/CYCB complexes. Activators of
endocycle
WEE1 Putatively involved in the inhibitory 1 1 2 1 1
phosphorylation of Tyr residues in CDKs,
though in CDKA;1 this phosphorylation
appears to be indispensible. Controls cell
cycle arrest in response to activation of the
DNA integrity checkpoint
CDC25 The sole catalytic domain of CDC25 3 1 1 2 1
phosphatase can be identified in plants, but
the N-terminal regulatory domain is
missing. It is debated but has not yet been
fully ruled out whether these CDC25 genes
indeed function to activate CDKs through
dephosphorylation of inhibitory Tyr
residues
(continued)
6 Cell Cycle Modules in Plants for Entry into Proliferation and for Mitosis 81

Table 6.1 (continued)

Core cell cycle genes Description Chlamydomonas Physcomitrella Selaginella Oryza sativa Arabidopsis
CDC20 The anaphase promoting complex (APC) is 1 5 2 3 6
responsible for the destruction of mitotic
substrates, it is activated at the metaphase/
anaphase transition by CDC20
CCS52 The CDH1 homologs are known as 1 4 2 2 3
CCS52A1 CCS52 genes in plants, they are required to
CCS52A2 establish the G1-phase state of low CDK
levels by activating the APC in telo- and
CCS52B
G1-phases. They positively regulate the
endocycle
CKS CDK-interacting protein related to yeast 1 1 2 1 2
SUC1; responsible for substrate binding.
Arabidopsis CKS is known to interact both
with CDKA and CDKB
RBR Related to animal retinoblastoma (RB), a 1 3 2 2 1
tumour suppressor which functions as a
transcriptional repressor for a broad
spectrum of genes. RBR is phosphorylated
and inactivated by the CDK/CYCD
complexes. RBR1 forms a co-repressor
complex involving other transcription
factors and chromatin modifying enzymes,
e.g. histone deacetylases. RBR is not only
involved in controlling proliferation, but
also in stem cell maintenance, cell
differentiation and imprinting; controls the
arrest of unfertilized gametophytes
E2F Related to animal E2F transcription factors, 1 3 2 4 3
E2FA pivotal targets for RB
E2FB
E2FC
DP Dimerization partners of E2Fs, required for 1 2 1 3 2
DPA their DNA binding
DPB
DEL DP/E2F-like proteins; can bind E2F 1 3 1 2 3
DEL1-3 elements as a monomer owing to their
tandem DNA binding domain. They do not
bind RBR. In Arabidopsis DEL1 inhibits
the endocycle by blocking the expression of
CCS52A genes
R1R2R3-MYB The R1R2R3-MYB type transcription 1 2 1 5 5
factors (TF) are conserved regulators of the
cell cycle both in animal and plant cells.
While in animals these TFs are known for
their role in G1/S, in plants they have been
shown to directly bind to the mitosis-
specific MSA element, and activate or
repress a suit of G2/M-specific genes
The name of core cell cycle genes are given in bold followed by the nomenclature of Arabidopsis subgroups. The number of homologous genes in
representative sequenced genomes is indicated. For more information and references see the main body of text as well as Van Leene et al. (2011).
Chlamydomonas genes are based on Bisova et al. (2005), Physcomitrella, Selaginella, Oryza sativa, Arabidopsis genes are taken from Banks et al.
(2011). R1R2R3-MYBs are defined in Haga et al. (2007) and Ito (2005). Missing orthologues were identified using the PLAZA resource for
comparative plant genomics

main mitotic cyclin, CDC13, with a single fusion protein and cell cycle regulatory system is the oscillation of CDK activ-
showed that this chimera molecule could carry out all CDK ity. This is mainly driven by the accumulation and degrada-
functions by deleting all the other remaining cyclins in this tion of the cyclin subunits. To experimentally demonstrate
organism (Coudreuse and Nurse 2010). At the heart of the this, they further engineered the CDC2-CDC13 fusion
82
molecule to become sensitive to inhibition by a “bulky” ATP
analogue, and showed that the absolute level of CDK activ-
ity was the only essential feature necessary to drive the three
key cell cycle transitions: a low level of CDK activity
triggers entry into S-phase, a medium level blocks the
repeated S-phase in G2 cells, while a burst of activity is
required for entry into mitosis. Whether in plants a single
CDK could carry out all cell cycle functions or the diverse
CDK and cyclin repertoire is indeed required to carry out
specialized functions has not yet been fully investigated.
To tune CDK activity so that it becomes switch-like to
flip the three main transition points, there are additional
layers of regulators incorporated into the central regulatory
system (Tyson and Novak 2008). Firstly, CDK activity can
be inhibited by a family of proteins that are related to the
animal CDK inhibitor protein KIP, named inhibitor of CDK
or KIP-related proteins (ICK/KRP). There are seven ICK/
KRP in Arabidopsis (De Veylder et al. 2007), but apparently
these proteins are missing in algae (Bisova et al. 2005;
Banks et al. 2011). It could be that only a small sequence
stretch is conserved in CDK inhibitors, and that might not be
recognised (Cross et al. 2011). In yeast and animal cells
these CDK inhibitory proteins mostly function in the G1 to
S-phase transition to build a bistable regulatory switch at the
transition point called start in yeast and restriction point in
animal cells (Tyson and Novak 2008). However, in plants it
has been proposed that ICK/KRP proteins might also func-
tion to establish the switch-like behaviour of the CDK oscil-
lator during the G2 to M-phase transition (Dissmeyer et al.
2010). This idea mostly came in to explain the void of
CDC25, the identity of which remains ambiguous in plants
(Dissmeyer et al. 2009). CDC25 is a phosphatase that
activates CDK by dephosphorylating conserved Thr14;
Tyr15 sites, its activity opposes WEE1 kinase, which
inhibits CDK activity through phosphorylation of the same
sites. Though these sites are conserved in plant CDKA
proteins, and WEE1 kinase is also present, the role of this
phosphorylation is unclear during normal cell cycle progres-
sion, as the wee1 mutant is indistinguishable from wild type,
and replacing wild type CDKA with a mutant form mimick-
ing the dephosphorylated state of CDKA has no effect either
on plant growth or development (Dissmeyer et al. 2009). The
authors therefore concluded that cell cycle control is effi-
ciently carried out without the reversible CDK phosphoryla-
tion in plants. To compensate for the missing negative
inhibitory loop built around CDK phosphorylation by
WEE1/CDC25 in animal and yeast cells, it has therefore
been suggested that the CDK inhibitors perform a similar
role to build the G2 to M-phase switch (Dissmeyer et al.
2010).
Based on mathematical modelling of the cell cycle regu-
latory system it was proposed that these additional layers of
negative regulators are fundamental to render unidirectional
Z. Magyar et al.
progression through all three main cell cycle control points
(Tyson and Novak 2008). This idea was also tested in fission
yeast by deleting the additional layers of CDK regulation.
Surprisingly, they could delete CDC25 and WEE1 without a
major effect on the yeast cell cycle progression (Coudreuse
and Nurse 2010). It is therefore not too surprising that in
plants the CDK phosphorylation is also dispensable for
normal cell cycle progression. Furthermore, they have also
deleted the yeast CDK inhibitor, RUM1 and shown that it is
dispensable for normal cell cycle progression. These results
demonstrate that a single monomolecular CDK molecule
without additional layers of regulators can sustain an effec-
tive mitotic cycle and thus constitutes the minimal architec-
ture of the eukaryotic cell cycle (Coudreuse and Nurse
2010). The expansion of CDK and cyclin families in animals
and plants could provide a more elaborate regulatory system
to meet more complex demands in multicellular organisms
(Fig. 6.1), but it appears that CDK subunits are also redun-
dant in metazoans (Hochegger et al. 2008).
How can we dissect the large complexity of cell cycle
regulatory systems in plants? There are 99 putative core cell
cycle genes in Arabidopsis, with CDKs and cyclins being
the largest families of proteins. Though this number is
somewhat lower in rice, 41, the number is still daunting
and makes the understanding of cell cycle regulation in
higher plants a complex task. To systematically map the
regulatory interactions among these core cell cycle
regulators in Arabidopsis, three independent methods have
been used in two studies, the yeast two hybrid (Y2H) assay,
the bimolecular fluorescence complementation (BiFC) assay
(Boruc et al. 2010), and affinity purification coupled to mass
spectrometry-based identification of proteins in these
complexes (AP–MS). Amalgamation of experimental data
on the binary links among the 58 core cell cycle proteins
generated by these three independent methods has uncov-
ered 416 interactions (Boruc et al. 2010; Van Leene et al.
2010; Van Leene et al. 2011). This dataset has been further
extended by literature-curated interactions and by those
computationally predicted based on the integration of multi-
ple sources of data such as sequence similarities, gene order
on chromosomes, and gene expression (Van Leene et al.
2011). Collectively, these works have established a
protein-protein interaction (PPI) network of the plant cell
cycle consisting of 506 binary interactions among 58
proteins. Besides being a valuable source of information to
help interpret existing experimental data and formulate new
hypotheses of the cell cycle PPI network, it has highlighted
the complex combinatorial interactions among CDKs,
cyclins and ICK/KRP proteins (Fig. 6.1). We can also
make some firm conclusions from the topology of the net-
work. CDKA is shown to make the most connections within
the network, emphasizing its central regulatory role,
although surprisingly this is almost exclusively restricted to
6 Cell Cycle Modules in Plants for Entry into Proliferation and for Mitosis
D-type cyclins and ICK/KRP proteins. The only exception to
this rule is the CDKA interaction with CYCA3-type cyclins.
D-type cyclins in association with CDKA have a predomi-
nant role in regulating entry into cell proliferation by phos-
phorylation of the retinoblastoma related (RBR) protein.
This protein controls the expression of a large battery of
cell cycle genes through the E2F transcription factors. Inter-
estingly, CYCA3-type cyclins share some of these properties
with D-type cyclins, such as the peak in expression at the G1
to S-phase boundary, and in association with CDKA they
promote the phosphorylation of RBR (Takahashi et al.
2010). Thus in plants CDKA seems to have become
specialized for regulating the entry into cell proliferation in
complex with CYCDs, CYCA3 and ICK/KRPs.
While in animals novel CDKs have evolved to fulfil G1 -
specific roles, plant lineage-specific CDKs, the B-type
CDKs, are functioning in the G2 to M-phases (De Veylder
et al. 2007). Consistent with a distinct role, the network
around B-type CDKs are clearly separated from those of
CDKA. B-type CDKs are associated with the mitotic
cyclins, CYCA1s, CYCA2s and CYCBs (Van Leene et al.
2010). The only exception to this rule is the interaction of
CDKB1;1 with CYCD4;1 which, when co-expressed, can
lead to ectopic cell division in epidermal cells (Kono et al.
2007) and initiation of lateral root formation (Nieuwland
et al. 2009). Since cyclins within the CDK complex have
important roles in determining substrate specificities,
CYCD4 might have retained broad substrate specificities
towards both the G1 to S and G2 to M targets. The SUC1
related CDK adaptor molecules, CKS1 and CKS2 also con-
nect to both CDKA;1 and CDKBs, further indicating some
possible share of substrates for these two modules.
If all KRPs are dedicated to control the CDKA;1/CYCD
complexes, which inhibitors regulate the mitotic CDKB
complexes? A prime candidate is the plant-specific CDK
inhibitor, SIAMESE (SIM), which was shown to antagonize
with CYCB and cooperate with the CYCB degradation fac-
tor, CCS52A1 (Kasili et al. 2010). A key decision to make
within the G2-phase of the cell cycle is whether to commit to
mitosis or engage in cycles of repeated DNA sysnthesis,
called endoreduplication or endocycle (see Maluszynska
et al. 2013; this volume). In yeast it was shown that blocking
of repeated DNA synthesis relies on the presence of CDK
activity in G2. In plants the onset of endocycle also involves
the selective inactivation of M-phase-promoting factors,
such as the B1-type CDK (CDKB1;1), through proteolytic
destruction of its cyclin partner, CYCA2;3 (Boudolf et al.
2009). In agreement, Arabidopsis relatives of the animal
fizzy-related activators of the anaphase promoting complex
(APC), CCS52A1 and CCS52A2, also promote the switch
from mitosis to endocycle (Larson-Rabin et al. 2009;
Vanstraelen et al. 2009). The anaphase-promoting complex/
cyclosome (APC/C) triggers the degradation of mitotic
cyclins leading to the exit from mitosis (Fulop et al. 2005).
83
Research into the regulation of the plant cell cycle is
largely motivated by its role in determining plant organ
size and potentially crop yield (Bogre et al. 2008). This
principally relies on the number of cells produced during
meristematic cell proliferation, and cell expansion after cells
have exited proliferation, a decision point that is tightly
controlled (Andriankaja et al. 2012). Can sequence variation
in cell cycle genes account for size differences among
plants? A large scale population genetics study of
polymorphisms in 61 core cell cycle genes across 30 natural
Arabidopsis accessions revealed signs of purifying selection
in central regulators, CDK subunits, WEE1 and RBR, large
effect mutations in CDKB1;1, adaptive protein evolution in
KRP6 and departure from equilibrium for CYCA3;3. These
data suggest that the robustness of the cell cycle regulatory
network is more due to functional redundancy than high
selective constraints (Sterken et al. 2009).
6.3 The Entry Module into the Cell Cycle
In plants growth is restricted to meristems, and thus a key
factor is the duration of cell proliferation and the timing of
the exit from proliferation to cell expansion and differentia-
tion (Doonan and Sablowski 2010). The current view is that
these events are controlled by an evolutionary-conserved
transcriptional regulatory switch, the E2F-RB pathway
(Inze and De Veylder 2006; Magyar 2008). The Retinoblas-
toma (RB) was the first tumour suppressor gene cloned from
mammalian cells, while the first adenovirus E2 binding
transcription factor (E2F) was identified based on its ability
to form a complex with the RB protein (van den Heuvel and
Dyson 2008). Originally E2F function was linked to cell
cycle control, but further studies revealed that E2F functions
extend beyond the control of cell proliferation. On the basis
of most recent animal studies E2F-RB may provide a
broadly utilised transcriptional switch for genes and thereby
co-ordinate the temporal expression of genes involved in the
regulation of cell cycle and differentiation (Korenjak and
Brehm 2005).
In principle, E2F works as a heterodimeric transcription
factor, forming a complex with a dimerization partner (DP)
protein prior to binding to the DNA and activating the
expression of target genes required for entry into the cell
cycle. In cells leaving mitosis, RB binds to E2F at
the carboxyl terminal RB binding motif and inhibits their
activities. This is converted, upon mitogen stimulation, by
hyperphosphorylation of RB by specific CDK-Cyclin D
complexes, leading to the activation of genes required for
DNA synthesis (Korenjak and Brehm 2005). In addition to
this repression function of RB through direct binding and
inhibition of E2Fs, RB can also function together with E2Fs
and actively repress transcription. This is because RB, and
its pocket protein relatives in mammalian cells p107 and
84
p130, are able to simultaneously bind to E2Fs and chromatin
remodelling enzymes such as histone deacetylases (HDACs)
(Rayman et al. 2002); or histone methyl transferases (e.g.
SUV39H1) (Liu et al. 2005). Animal E2Fs have been classi-
fied as activators and repressors based on whether they
transactivate genes which are repressed by RB or form a
co-repressor complex with RB, respectively (van den
Heuvel and Dyson 2008). However, recent in vivo studies
have clearly demonstrated that activator E2Fs can also work
as repressors and, vice versa, repressor E2Fs can also func-
tion as activators in certain developmental contexts and in a
tissue-specific manner (Danielian et al. 2008; Infante et al.
2008; Tsai et al. 2008; Chen et al. 2009; Chong et al. 2009;
Wenzel et al. 2011).
Initially E2F function was linked to the control of DNA
synthesis through regulating the expression of critical cell
cycle genes required for S-phase progression. However, in
Drosophila it was shown that dE2F and RB (called RBF1-2
in the fly) proteins can control replication through a mecha-
nism which is distinct from their effects on gene expression
(Cayirlioglu et al. 2001, 2003). Although the exact mecha-
nism is still unclear, dE2F and RB proteins were found to
associate with origin recognition complex (ORC) proteins
and they were co-localized at amplifying loci (Royzman
et al. 1999; Bosco et al. 2001).
Several studies have revealed that animal E2Fs function
not only in the control of the G1/S transition but are also
involved in the regulation of the G2/M transition. A number
of critical mitotic genes in animal cells have been identified as
E2F targets including cyclin B1, the regulatory subunit of
CDK1 in mammalian cells (Zhu et al. 2005), CDC25 phos-
phatase, an activator of the mitotic CDK1 in Drosophila
(Neufeld et al. 1998), and MAD2, a mitotic checkpoint com-
ponent gene in mouse (Hernando et al. 2004). Interestingly,
the regulation of MAD2 expression by RB/E2F transcrip-
tional regulators was found to play important roles in the
control of chromosome stability (Sage and Straight 2010).
Therefore it is suggested that E2F-RB may mediate both the
accurate and timely replication of DNA and the accurate
distribution of chromosomes during mitosis (Bosco 2010).
Moreover, studies in animal cells have demonstrated that
the E2F-RB repressor complexes are present in actively
dividing cells and regulate both the expression of genes
involved in differentiation and the G2- and M-phase transi-
tion of the cell cycle (Dimova et al. 2003; van den Heuvel
and Dyson 2008). These newly identified E2F-RB com-
plexes are regulated differently from the classical E2F-RB
complexes since they are insensitive to the traditional RB-
kinases consisting of cyclin D and CDK proteins.
The E2F-RB network is remarkably conserved between
the plant and animal kingdoms. E2F and RB homologs
have been identified from the unicellular green alga
Chlamydomonas reinhardtii to the higher plants including
Z. Magyar et al.
the model plant Arabidopsis thaliana (Inze and De Veylder
2006). Interestingly, nearly 25 % of the Arabidopsis genes in
total contain consensus E2F binding element(s) within their
promoter region, indicating that E2Fs could play a funda-
mental role in regulating many aspects of plant development
including cell proliferation and differentiation (Vandepoele
et al. 2005, 2009). Arabidopsis contains a family of six E2F-
related proteins (Vandepoele et al. 2002). Three of them,
E2FA, E2FB and E2FC, are structurally related to the canon-
ical animal E2Fs since they all have the conserved domains
characteristic for the animal E2F1-3, including the DNA-
binding, dimerization, transactivation and RB binding dom-
ains. The three other E2F-related members in Arabidopsis are
DEL1, DEL2 and DEL3 (DP-E2F-like proteins). These are
structurally related to mammalian E2F7 and E2F8 that lack
all the E2F-specific domains except the DNA-binding
domain, which is present in tandem duplication. Arabidopsis
DELs represent a subgroup of the E2F family which does not
require heterodimer formation binding to the DNA with the
two known dimerization partner proteins, DPA and DPB, and
their function is not directly regulated by RBR1 protein since
they lack the RB-binding motif. In analogy to animal
systems, plant E2Fs have also been classified as transcrip-
tional activators (E2FA and E2FB), or transcriptional
repressors (E2FC), although most of these data were derived
from over-expression studies (Magyar 2008). Genome wide
expression profiling has shown that ectopic co-expression of
E2FA with the dimerization partner A (DPA) not only leads
to transcriptional activation but also to the transcriptional
repression of a large number of genes, perhaps indicating
that the same E2F/DP complex can be an activator of some
genes and a repressor of others (Vandepoele et al. 2005).
Functional characterization of the individual members of
Arabidopsis E2Fs has already revealed differences among
them: ectopic expression of E2FA with DPA resulted in
strong activation of both the mitotic cell cycle and endocycle
(De Veylder et al. 2002; Kosugi and Ohashi 2003),
overexpression of E2FB was also able to activate mitosis
but it repressed the endocycle (Magyar et al. 2005; Sozzani
et al. 2006), whereas reduction in the level of E2FC con-
firmed its negative regulatory function in mitosis but a
positive one in endoreduplication (del Pozo et al. 2006).
Thus, E2FB and E2FC are antagonistic transcription
factors, while E2FA can regulate both proliferation and
endocycle. The target gene specificities of E2FB and E2FC
are not fully understood, but the regulation of the mitotic
CYCB1;1 gene is clearly opposite for E2FB and E2FC (del
Pozo et al. 2006; Sozzani et al. 2006). Lowering the amount
of E2FC by RNAi markedly upregulated CYCB1;1 expres-
sion, much more than the other E2F target genes such as
CDC6, EXP3, which have clear E2F binding elements, while
CYCB1;1 does not. This suggests that CYCB1;1 might be a
direct target for E2FC-dependent repression. Curiously,
6 Cell Cycle Modules in Plants for Entry into Proliferation and for Mitosis
E2FC-RNAi only had an effect on CYCB1;1 expression in
mature leaves but not in young leaves, suggesting that E2FC
forms a repressor complex in differentiated cells. The
endocycle was also compromised in the leaves of E2FC-
RNAi plants, suggesting that the repression of mitotic genes
such as cyclin B1;1, by E2FC is part of the switch from
mitotic cell cycle to endocycle. Curiously, E2FC appears to
repress mitosis in mature leaves but it is most abundant in
proliferating cells (del Pozo et al. 2002, 2006). In accordance
with these data from Arabidopsis, in Drosophila embryos
the expression of cyclin B1 and B3 genes are also repressed
as cells enter endocycle (Edgar and Orr-Weaver 2001).
Trichome development is linked with endocycle, but by
targetting the expression of the mitotic cyclin B1;2 to
trichomes, the endocycle is converted into mitosis, leading
to multicellular trichomes (Schnittger et al. 2002). E2FC
might function as a transcriptional repressor on E2F target
genes. This is supported also by the over-expression of a
stabilized mutant DE2FC lacking the amino terminal in
Arabidopsis which in dark grown plants led to the repression
of CDC6, a gene involved in S-phase (del Pozo et al. 2002).
In mouse, there are two alternative splicing variants of E2F3,
a full length form (E2F3a) and an amino terminally deleted
(E2F3b) form. These two variants have opposite functions;
E2F3a is an activator while E2F3b is a repressor (Leone
et al. 2000; Aslanian et al. 2004). The function of the N-
terminal part of E2F3 is unknown but in E2F1 it is required
for ubiquitin-mediated degradation (Marti et al. 1999). In
plants the N-terminal extension of E2Fs has numerous CDK
phosphorylation sites. For E2FC this N-terminal part was
shown to be required for ubiquitin-mediated degradation.
Deletion of the N-terminal part led to the stabilization of
E2FC and E2FA (del Pozo et al. 2002; Magyar et al. 2005).
Over-expression of the N-terminally deleted DE2FC mutant
together with DPB led to growth arrest due to compromised
cell proliferation and an elevated endocycle (del Pozo et al.
2006), providing further evidence that E2FC represses mito-
sis but promotes endocycle. Genome wide gene expression
data with plants overexpressing E2FA-DPA or E2FC-DPA
revealed minimal overlap. Whether the target specificity is
also determined by the dimerisation partner, DPA or DPB
remains to be established. While E2FC represses CYCB1;1,
over-expression of E2FB, or co-over-expression of E2FA
with DPA activates it. Whether this opposing function of
E2FC and E2FB is through competition of these two factors
for the same site or through alternative E2F-binding sites or
whether these E2Fs indirectly regulate CYCB1;1 expression
is not known. However, it has been established that E2FB
and E2FC amounts are oppositely regulated by light
conditions. In the dark E2FC amounts are high compared
with E2FB, while in the light it is the opposite, and these
depend on light-signalling components, DET1 and COP1
(Lopez-Juez et al. 2008; Berckmans et al. 2011a). E2FC
85
was also shown to be destabilised in light through the
ubiquitin-SCF pathway (del Pozo et al. 2002). The atypical
E2F, DEL1 is the target for E2FB and E2FC and these two
antagonistically regulate DEL1 levels through competition
for a single E2F cis-acting binding site (Berckmans et al.
2011a). DEL1 in turn regulates CCS52As and entry into
endocycle in response to light conditions (Lammens et al.
2008). The ratio between E2FC and E2FB could also be
influenced by auxin. Auxin concentration has a dose-
dependent effect on cell division and cell elongation; high
auxin promotes cell proliferation, while low auxin leads to
exit from the cell cycle and stimulates cell elongation
(Scheres and Xu 2006). Auxin-dependent reactivation of
E2FA was shown to be an important factor for the asymmet-
ric cell division during lateral root initiation (Berckmans
et al. 2011b). Auxin was also shown to stabilise E2FB,
and co-over-expression of E2FB with DPA could sustain
proliferation in the lack of auxin (Magyar et al. 2005).
Cells in the E2FB-DPA plants were extremely small
suggesting that E2FB inhibits growth. Over-expression of
E2FB alone in Arabidopsis plants also led to overproli-
feration, small cells and these plants had short roots (Sozzani
et al. 2006). A possible model that can be derived from
these data is that elevated levels of E2FB maintain prolifer-
ation in the meristems until the E2FC level rises above E2FB
in mature leaves leading to the switch from mitotic cell cycle
to endocycle through the repression of mitotic genes
(Fig. 6.2).
All these data underline the importance of plant E2Fs in
regulating the G2 to M-phase transitions in a similar way to
that found for animal E2Fs (Neufeld et al. 1998; Hernando
et al. 2004). Elevated E2FB levels in BY-2 tobacco cell lines
resulted in a shortened cell cycle which is analogous to what
was found for Drosophila dE2F1 that in parallel controls two
important regulators for the G1 to S and G2 to M-phase
transitions (Magyar et al. 2005). E2FB targets the promoter
of CDKB1;1 a critical regulator for the G2 to M transition
(Magyar and Bogre, unpublished result).
E2FA could be at the top of the hierarchy of the E2F
regulatory network. It does not contain an E2F element in its
own promoter but has been shown to activate the expression
of both E2FB and E2FC (Vandepoele et al. 2005). Plants
over-expressing both E2FA and DPA are severely stunted
(De Veylder et al. 2002; Kosugi and Ohashi 2003; Magyar
et al. 2012); cells in some tissues show over-proliferation
while in others over-endoreduplication. E2FA may also
repress cell proliferation in mature leaves in a similar way
to that shown for E2FC (He et al. 2004). E2FA is most
abundant in meristems and specifically peaks in S-phase
cells, but can also be detected in endoreduplicating cells.
Transcriptomics analysis of an E2FA/DPA line showed a
strong up-regulation of genes involved in DNA synthesis
(e.g. CDC6, ORC1, CDC45, RNRII, MCM3), but genes with
86
Fig. 6.2 Regulation of cell proliferation versus endocycle and differ-
entiation by the RBR1/E2F transcriptional regulatory switch. RBR1
phosphorylation is regulated by sucrose availability dependent on the
CYCD3;1 and KRP2. The released E2FB is driving cells into prolifer-
ation. Auxin and light regulate proliferation by oppositely affecting the
activator E2FB and repressor E2FC levels. Opposite to E2FB, elevated
sucrose levels increase the association of RBR1 with E2FA, which
roles in the G2 to M-phase transition, such as cyclin B1;1,
and CDKB1;1 were also upregulated (Vlieghe et al. 2003;
Vandepoele et al. 2005). CDKB1;1 was shown experimen-
tally to be a direct target of the E2FA-DPA heterodimer
(Boudolf et al. 2004a). The paradox is how E2FA promotes
the G2 to M transition when its expression is restricted to the
S-phase and how it can regulate the two antagonistic pro-
cesses, endocycle and G2 to M transition. Recently it was
shown that in proliferating cells E2FA forms a stable com-
plex with RBR1 to repress genes involved in endocycle and
differentiation, in this way the E2FA-RBR1 complex
maintains the competence for cell proliferation in the meri-
stem (Magyar et al. 2012). E2FA over-expression leads to
elevated levels of the E2FA-RBR1 complex and increased
proliferation. However, outside the meristem E2FA, by
some unknown mechanism, dissociates from RBR1 and
can stimulate endocycle (Magyar et al. 2012).
In Arabidopsis, there is only a single RBR1 protein. How
RBR1 functions through the three distinct E2Fs, the timing
and regulation of their interaction and the repression of
distinct batteries of genes by these different E2F-RBR1
complexes is not well understood. RBR1 protein is most
abundant in proliferating cells (Wildwater et al. 2005;
Borghi et al. 2010; Umbrasaite et al. 2010).
The first identified rbr1 mutant allele was found to be
non-viable, with defects during both male and female game-
togenesis due to over-proliferation, suggesting a repressive
function for RBR1 in the mitotic cell cycle (Ebel et al. 2004).
Z. Magyar et al.
leads to the repression of genes involved in endocycle. In this way
the E2FA-RBR1 complex contributes to the maintenance of meristems
by the inhibition of cell differentiation. Later on RBR1 dissociates from
E2FA by an unknown mechanism, and RBR1-free E2FA stimulates
endocycle in differentiated cells. Arrows indicate activation, hammers
repression, white arms are stimulatory, black arm indicate inhibitory
inputs
RBR1 is also required for cell fate determination in
gametophytes. Reduction in RBR1 amounts in somatic
tissues also leads to over-proliferation and delayed differen-
tiation (Park et al. 2005; Desvoyes et al. 2006). In addition,
RBR1 has been shown to play a role in repressing endocycle
and maintaining genome integrity (Henriques et al. 2010;
Johnston et al. 2010; Magyar et al. 2012).
Arabidopsis RBR1 has been shown to be important for
stem cell maintenance which relies on the canonical CYCD/
RBR/E2F pathway; reducing RBR1 amounts by RNAi or
elevated RBR1 phosphorylation through the over-expression
of CYCD3;1 produced extra stem cell layers while over-
expression of RBR1 or hypophosphorylation induced by
increased amounts of KRP2 led to the loss of stem cells.
In accordance with the canonical model, having more
RBR1-free E2Fs in cells by over-expression of both E2FA
and DPA also produced more stem cells (Wildwater et al.
2005).
RBR1 over-expression stimulates early differentiation
both in shoot and root meristems (Wildwater et al. 2005;
Wyrzykowska et al. 2006). Besides CYCD/CDKA, there are
also other signalling pathways that can control the transcrip-
tional activity of the E2F-RBR1 complex. It was found that
the mutation or silencing of the two ribosomal protein S6
kinase genes in Arabidopsis led to proliferation in growth
limiting conditions such as lack of sucrose (Henriques et al.
2010). This deregulation in proliferation also resulted in
frequent aneuploidisation. S6 kinase 1 was shown to interact
6 Cell Cycle Modules in Plants for Entry into Proliferation and for Mitosis
with RBR1 and this interaction promotes the nuclear
localization of RBR1. How other signalling pathways, such
as the mitogen-activated protein kinase pathway, or Aurora
kinase can regulate E2F-RBR1 functions remains to be
established.
6.4 Transcriptional Regulation at G2
Phase to Mitosis and the Onset
of Endoreplication
The most established function for E2F/DP is to control
transcription during the G1 to S-phase transition by
activating genes required for DNA replication and repair,
thereby generating waves of gene expression peaking at this
stage. The E2F transcription factors might also regulate the
following waves of gene expression at G2, which may be
associated with progression through G2 and entry into mito-
sis. There are a relatively small number of genes that show
this pattern of expression, they include plant-specific B1-
type CDKs (CDKB1) and A2-type mitotic cyclins
(CYCA2); (Menges et al. 2003, 2005). In Arabidopsis,
E2FA activates the transcription of CDKB1;1 which has
E2F binding sites in its promoter (Boudolf et al. 2004a).
Both CDKB1;1 and CYCA2;3 are known to be involved in
the onset of endoreduplication during organ development in
Arabidopsis (Boudolf et al. 2004b; Imai et al. 2006). It has
been proposed that these proteins form an active complex
that maintains mitotic division and represses endoredu-
plication onset (Boudolf et al. 2009). There is no evidence
that CYCA2;3 is also directly regulated by E2FA, instead a
report shows that it is regulated by INCREASED LEVEL
OF POLYPLOIDY1 (ILP1), whose loss-of-function resulted
in reduced polyploidy and upregulation of all CYCA2
members (Yoshizumi et al. 2006). The authors concluded
that this putative transcription factor may repress CYCA2
transcription, thereby promoting exit of mitotic division and
onset of endoreplication. Further genome-wide studies are
needed to understand if ILP1 acts as a master regulator of
G2-phase genes by repressing other genes that are expressed
at this stage of the cell cycle.
6.4.1 Genes Expressed During Late G2
and Mitosis
Following the peak of CDKB1 and CYCA2;3 expression at
G2, a different group of genes is induced shortly before entry
into mitosis. This group contains a much larger number of
genes including mitotic cyclins of A1, B1 and B2 types
(CYCA1, CYCB1 and CYCB2) and plant-specific B2-type
CDKs (CDKB2); (Menges et al. 2005; Kato et al. 2009).
Besides these core cell cycle genes with presumable mitotic
87
function, this group also contains many genes acting for
spindle checkpoint (e.g., CDC20.1, MAD2, and BUB3),
dynamics of mitotic microtubule machinery (many kinesins
and other microtubule-associated proteins), and genes
involved in cell plate formation (e.g., KNOLLE, HINKEL/
AtNACK1, and PLEIADE). We have recently defined
185 genes showing such G2/M-specific expression in
Arabidopsis (Haga et al. 2011). Most of these genes com-
monly contain promoter motifs similar to the MSA element,
this has previously been identified as a cis-acting element
important for G2/M-specific transcriptional activation in
tobacco cells (Ito et al. 1998). The MSA element may be
involved in evolutionarily conserved mechanisms for G2/M-
specific promoter activation, because this motif is commonly
found in G2/M-specific genes both in dicots, monocots as
well as in moss.
6.4.2 Transcriptional Activation of
G2/M-Specific Genes
Several lines of evidence suggest that R1R2R3-type MYB
transcription factors directly bind to the MSA element, and
regulate a suit of G2/M-specific genes (Ito 2005). These
types of MYB transcription factors are broadly conserved
among eukaryotes and may be prototypes of the diverse
R1R2R3-type MYB proteins found in plants. R1R2R3-
MYB proteins are structurally subdivided into at least four
groups which we call A-, B-, C- and D-type MYBs. Among
them, A-type MYBs are conserved in angiosperms and act as
transcriptional activators (Fig. 6.3). In transient expression
assays in BY-2 protoplasts, tobacco A-type MYB proteins,
NtMYBA1 and NtMYBA2, can increase promoter activity
of G2/M-specific genes in an MSA-dependent manner (Ito
et al. 2001). It has been shown that NtMYBA2 has a negative
regulatory domain in its C-terminus whose deletion
enhances the activity of NtMYBA2 for transactivation of
G2/M-specific genes (Araki et al. 2004). Microarray analysis
showed that over-expression of the hyperactive form of
NtMYBA2 lacking the negative regulatory domain resulted
in up-regulation of many G2/M-specific genes in BY-2 cells
(Kato et al. 2009). Conversely, simultaneous mutation of the
two A-type MYB genes, MYB3R1 and MYB3R4, resulted in
the down-regulation of many G2/M-specific genes in Arabi-
dopsis (Haga et al. 2011). The most prominent defect caused
by the double myb3r1 myb3r4 mutation is the frequent
occurrence of incomplete cytokinesis during somatic cell
division. In agreement with the proposed roles of A-type
MYB, this cytokinetic defect is mainly due to reduced
expression of KNOLLE that is essential for cell plate forma-
tion during cytokinesis (Haga et al. 2007).
Cellular activity of A-type MYB proteins is regulated
both at the transcriptional and post-transcriptional level
88
Fig. 6.3 Transcriptional regulation of G2/M-specific genes. R1R2R3-
type MYB transcription factors directly bind to the MSA element, and
regulate a suit of G2/M-specific genes. A-type MYBs (MYB3R4 and
MYB3R1) are transcriptional activators and they positively regulate
many G2/M-specific genes. The RING E3 ligase, encoded by
HISTONE MONOUBIQUITINATION1 (HUB1) is also involved in
regulating the G2/M-specific gene transcription. Whether HUB1
directly regulates promoter activities or acts through the R1R2R3-
type MYB transcription factors is not yet known. Opposing these
positive regulators is the E2FC transcription factor. Whether E2FC is
part of a larger complex together with R1R2R3-type MYBs remains to
be established. Arrows indicate activation, hammers repression, black
lines are experimentally proven and grey lines are hypothetical
during the cell cycle, such that transcription of their target
genes is oscillating, with maximum levels at G2/M. In
tobacco cells, NtMYBA2 shows cell cycle-regulated expres-
sion peaking at G2/M, contains an MSA element in its
upstream region, and is up-regulated by the hyperactive
form of NtMYBA2 (Ito et al. 2001; Kato et al. 2009). This
suggests that transcription of NtMYBA2 is activated by
itself in a positive feedback loop. Similarly, NtMYBA2 is
directly phosphorylated and activated by a CDK in complex
with CYCB1, which is a potential target being transcription-
ally activated by NtMYBA2 (Araki et al. 2004). All these
data suggest the presence of positive feedback loops in
NtMYBA2 activation at transcriptional and post-trans-
criptional levels, which may enable the rapid increase of
the regulatory proteins in the narrow window of the cell
cycle.
6.4.3 Transcriptional Repression
of G2/M-Specific Genes
In addition to A-type MYB transcriptional activators,
repressor-type R1R2R3-MYB proteins also exist and partic-
ipate in the regulation of G2/M-specific genes. The B-type
MYB protein, NtMYBB when transiently over-expressed in
tobacco BY-2 cells, was shown to repress the G2/M-specific
Z. Magyar et al.
genes CYCB1 and NACK1, which was dependent on the
presence of an MSA-element in their promoters (Ito et al.
2001). Replacement of the C-terminal region of NtMYBB
with VP16 activation domain converted this transcriptional
repressor into a strong activator. This suggests that the
function for transcriptional control is determined by C-
terminal regions and not by the N-terminally located MYB
domain that is responsible for recognition of the MSA
sequence. When NtMYBA2 and NtMYBB were simulta-
neously over-expressed, NtMYBB could decrease promoter
activity that was enhanced by NtMYBA2, suggesting that
NtMYBB may act as a competitive repressor (Ito et al.
2001). Therefore, G2/M-specific transcription may be
regulated by a dynamic balance between an activator
(NtMYBA2) and a repressor (NtMYBB), both of which
bind to the same MSA sequence. NtMYBB is expressed
constitutively throughout the cell cycle, in contrast to the
G2/M-specific expression of NtMYBA2. Based on the
findings from R1R2R3-MYB genes, a model was proposed
that G2/M-specifc genes are normally repressed by constitu-
tive expression of NtMYBB, whereas their transcription
is triggered when activity of NtMYBA2 increases and
overcomes NtMYBB-mediated repression. NtMYBB is
also expressed in non-dividing cells, but it remains to be
determined if this MYB protein is also involved in the
maintenance of the repressive state of G2/M-specific genes
in quiescent cells.
A recent report suggests that E2F transcription factors
may also play a role in repressing G2/M-specific genes.
E2FC, one of the three E2F proteins in Arabidopsis, is
regarded as a transcriptional repressor because it does not
have transcriptional activation properties in transient expres-
sion assays, and its over-expression causes down-regulation
of CDC6, a representative E2F target (del Pozo et al. 2002).
RNAi-mediated knockdown of E2FC resulted in extra cell
divisions during leaf development with reduced levels of
endoreplication (del Pozo et al. 2006). In the E2FC knock-
down there is a significant up-regulation of S-phase genes in
mature leaves but not in young leaves. This suggests that a
primary role of E2FC is to establish or maintain the
repressive state of S-phase genes during or after cessation
of cell divisions in organ development. In addition, a marked
increase of CYCB1;1 transcript was also found in mature but
not young leaves of E2FC knockdown plants (del Pozo et al.
2006). Conversely, over-expression of E2FC diminished
expression of the CYCB1;1-GUS transgene and the endoge-
nous KNOLLE gene. Consistently, E2FC is expressed in
mature organs comprising mostly non-dividing cells, such
as cotyledons and mature leaves, but as stated above, it is
most abundant in meristematic tissues in Arabidopsis (del
Pozo et al. 2002). One attractive hypothesis is that G2/M-
specific genes are regulated positively by MYB3R1/4 and
negatively by E2FC, and that their actions are dependent on
6 Cell Cycle Modules in Plants for Entry into Proliferation and for Mitosis
the stage of organ development (Fig. 6.3). It would be
interesting to examine if E2FC knockdown also affects
other G2/M-specific genes in microarray analysis, and if
such effects are dependent on the MSA element or not.
6.4.4 Other Factors Affecting the
G2/M-Specific Transcription
Among G2/M-specific genes, CYCB1;1 has been extensively
studied as a representative gene of this group. Initial genetic
approaches identified a mutation which affected GUS
reporter expression driven by the CYCB1;1 promoter in
Arabidopsis, but that mutation has not yet been mapped
(Himanen et al. 2003). Putative regulatory proteins for
CYCB1;1 transcription have been identified by a different
strategy. Two putative transcription factors, the ELM2
domain-containing protein and transcription elongation fac-
tor TFIIS family protein, were isolated in a protein complex
that binds to affinity beads covalently attached to an oligo-
nucleotide of the MYB binding sequence in the CYCB1;1
promoter (Planchais et al. 2002). While in a more recent
study, TCP20, a TCP family transcription factor, has been
shown to bind to the GCCCR element found in CYCB1;1 and
some ribosomal protein genes (Li et al. 2005). This element
is required for high level transcription from the CYCB1;1
promoter in the presence of the MSA element. Future reverse
genetic studies are needed to confirm if these factors are
actually required for transcription of CYCB1;1 and other
G2/M-specific genes.
It has been recently proposed that chromatin modifications
are involved in the G2/M-specific transcription in Arabidopsis.
The RING E3 ligase encoded by HISTONE MONOUBIQUI-
TINATION1 (HUB1) has been identified as a gene responsible
for reduced leaf size in ang4 mutants (Fleury et al. 2007).
HUB1 is an ortholog of BRE1 from yeast and human which
carries out monoubiquitination of histone H2B, a posttransla-
tional modification important for transcriptionally active chro-
matin. The reduced leaf size in ang4/hub1 mutants may be
caused by increased cell cycle duration, which is, in turn,
caused by the downregulation of many G2/M-specific genes.
Microarray analysis of shoot meristems from the ang4/hub1
mutant showed significant down regulation of 66 out of 82
mitosis-specific genes defined by Menges et al. (2005), most of
which contain the MSA motif in their promoter regions. This
suggests that HUB1 may positively and selectively affect
transcriptional activity, and that such selective action for
G2/M-specific genes may be achieved by a mechanism that
recognizes the MSA element. One likely explanation is that
HUB1 may be recruited to the MSA sites with the aid of
R1R2R3-MYB transcription factors that have MSA-binding
ability. In such a scenario, transcriptional activation regulated
by MYB3R1/4 may be mediated through chromatin
89
modifications, i.e., H2B monoubiquitination, around the
MSA sites during mitosis (Fig. 6.3). It remains to be
investigated if G2/M-specific genes are actually
monoubiquitinated, when such modification occurs in the
cell cycle, and if the effects of the ang4/hub1 mutation are
dependent on MYB3R1/4.
6.5 Spatial Organization of Mitosis
Microtubules are required for cellular transport, they serve
as retention devices for many signalling molecules, and are
used for the construction of the chromosome segregation
machinery, the mitotic spindle. Microtubule assembly and
orientation is directed from specialized structures such as the
spindle pole bodies in fungi and centrosomes in animal cells.
Centrosomes are known to organize both the interphase
microtubules and the bipolar mitotic spindle in animal
cells, where their function is modulated by phosphorylation.
Defects of centrosomes result in chromosome instability
(Kramer et al. 2011). The major mitotic kinases are the
cyclin dependent kinase1/cyclinB complex, Polo-like kinase
(Plk) and the Aurora kinases. These mitotic kinases are
known to control the spatial and temporal events partially
through the phosphorylation of centrosomal proteins
(Ma and Poon 2011).
The microtubule network is made up of the conserved
tubulin family, commonly through the polymerization
of a-tubulin and b-tubulin. g-Tubulin, in its soluble cyto-
plasmic state forms small complexes with two additional
g-tubulin complex proteins (GCPs), GCP2 and GCP3.
g-Tubulin is also present as a large ring complex (gTuRC)
that is built of small tetrameric g-tubulin complexes and that
is established as a core unit needed for microtubule
nucleation from centrosomes. Other specific components of
the large gTuRCs, GCP4–6 are not essential for nucleation
of microtubules but increase nucleation efficiency (Raynaud-
Messina and Merdes 2007). However, non-centrosomal
nucleation of microtubules occurs not only in acentrosomal
cells such as oocytes but also in cells equipped with
centrosomes (Bartolini and Gundersen 2006), and the role of
g-tubulin in the alternative pathway of microtubule nucleation
is generally accepted (Janson et al. 2005; Schuh and Ellenberg
2007). Discrete structures for microtubule nucleation, such as
centrosomes, are absent in higher plant cells and non-
centrosomal microtubules are nucleated from dispersed g-
tubulin positive sites in the cytoplasm, with membranes, and
with pre-existing microtubules (Murata et al. 2005; Binarová
et al. 2006; Pastuglia et al. 2006). Localization of g-tubulin
around the outer nuclear envelope and its association with
pre-kinetochores and kinetochores in early mitosis may
depend on phosphorylation by mitotic kinases (Binarová
et al. 1998, 2000). Furthermore, g-tubulin together with the
90
nuclear pore complex Nup107-160, are also localized to
kinetochores of mitotic HeLa cells (Mishra et al. 2010),
and thus it appears that g-tubulin has a conserved function
in the nucleation of kinetochore microtubules in the early
stages of mitosis both in plant and animal cells. Apart from
its association with kinetochores, g-tubulin is also present
along plant spindle microtubules where it shows gradual
translocation from the kinetochore region to the
acentrosomal poles during mitotic progression (Liu et al.
1993; Dryková et al. 2003). Correct localization of g-tubulin
to mitotic spindle microtubules is necessary for the organi-
zation of acentrosomal spindles in Drosophila oocytes, where
g-tubulin stabilizes the attachment of spindle microtubules at
the kinetochores (Hughes et al. 2011). These data suggest
that the precise spatial regulation of g-tubulin localization
throughout the cell cycle is required to ensure g-tubulin
functions in both centrosomal and acentrosomal cells. Com-
pared with the extensive data available on the regulation of
cell cycle specific centrosomal function, the regulation of
dispersed centrosomes in acentrosomal cells is much less
understood.
Spin down experiments with taxol-polymerized plant
microtubules confirmed the presence of g-tubulin and Cyclin
Dependent Kinase A (CDKA) with microtubules (Wein-
gartner et al. 2001; Dryková et al. 2003). Besides CDKA,
other molecules that make up the core of the cell cycle
machinery were also shown to be associated with
microtubules by mass spectrometry-based proteomics of
taxol-stabilised microtubule pellets (unpublished data;
Jones, Binarová, Bogre). Centrosome-independent nucle-
ation of spindle microtubules requires augmin (Goshima
et al. 2008) and it assembles into a complex with g-tubulin,
dependent on Cyclin Dependent Kinase1 (CDK1), Polo
kinase and Aurora signalling (Johmura et al. 2011). Augmin
related proteins are important for spindle and phragmoplast
organization in plants (Ho et al. 2011). It has been shown
that g-tubulin and GCPs are regulated in a cell cycle depen-
dent manner by phosphorylation. Analysis of cell cycle
phosphoproteome of yeast spindle pole bodies revealed
dynamic phosphorylation of g-tubulin and GCPs during
cell cycle progression (Keck et al. 2011). Clustering of
phosphosites that create charged regions were prominent
within yeast centrosomal proteins and suggestive of phos-
phorylation-mediated protein-protein interactions (Schw-
eiger and Linial 2010). Two G1-phase specific and six
M-phase specific phoshosites are present on the g-tubulin
molecule and these are highly conserved in eukaryotes
(Keck et al. 2011). It has been shown in two independent
studies that the mutation of a single CDK phosphorylation
site, Tub4 S360D, causes mitotic delay and aberrant ana-
phase spindle elongation (Keck et al. 2011; Lin et al. 2011).
This phosphosite is conserved on plant g-tubulin and
suggests that CDK phosphorylation may also control plant
Z. Magyar et al.
g-tubulin function in plants. Our analysis of the
co-expression neighbourhood with g-tubulin uncovered
CDKB2.1. It would be interesting to examine experimen-
tally, whether CDKB2.1 is involved in phosphoregulation of
g-tubulin (Fig. 6.4).
g-Tubulin and GCP3 protein have been identified to be
amongst the targets of Aurora signalling in mammalian cells
(Sardon et al. 2010). Aurora kinases are central to the coordi-
nation of many cellular events of cell division and differenti-
ation. There are three aurora kinases in animal cells,
AURORA A–C. AURORA A localizes to centrosomes and
along mitotic spindle microtubules, and plays a role in cen-
trosome maturation, spindle pole organization and mainte-
nance in metazoan cells. Depletion of Aurora A delays
chromosome condensation and CDK1 activation (Liu and
Ruderman 2006). Aurora B, as a chromosomal passenger, is
essential for normal chromosome segregation and cytokine-
sis (Carmena and Earnshaw 2003). As in animal cells, three
Aurora kinases have been identified in the Arabidopsis
genome, named as AtAurora1, 2, and 3. Meristem-defective
phenotypes observed in RNAi plants with silenced Aurora
kinases suggest they play a role in maintaining meristematic
cells in the mitotic cycle (Petrovská et al. 2012). A higher
level of endoreduplication occurs in plants with
downregulated Aurora kinases, demonstrating that Aurora
kinases may interconnect cell cycle signalling with unknown
targets involved in the switch from cell division to differenti-
ation. In mammalian cells Aurora B phosphorylates retino-
blastoma (RB) protein in vitro and in vivo at serine 780.
Inhibition of Aurora B led to RB hypophosphorylation
accompanied by endoreduplication (Nair et al. 2009). There-
fore Aurora B kinase directly regulates the RB protein and
the postmitotic checkpoint by preventing endoreduplication
in cells with aberrant mitosis.
The AtAurora1 kinase localizes with the prophase spindle
around the nuclei, with the preprophase band of cortical
microtubules, and with mitotic spindle microtubules
(Demidov et al. 2005, 2009). AtAurora1 physically interacts
with its activator the AtTPX2 protein, and the co-
localization of both proteins on preprophase spindle
microtubules suggests that active AtAurora1 kinase is
involved in g-tubulin-driven microtubular nucleation during
plant mitosis (Petrovská et al. 2012). The AtAurora1 kinase
and TPX together with g-tubulin localize with a specific
subset of early phragmoplast microtubules adjacent to chro-
matin and distal to the cell plate e.g., at the site where
phragmoplast microtubules are nucleated. Activity of
Aurora A kinase is required for nucleation of microtubules
with or without centrosomes (Tsai and Zheng 2005). Pres-
ence of AtAurora1 and its activating subunit AtTPX2 with g-
tubulin on microtubules suggests a link between the Aurora
kinases and g-tubulin mediated processes, such as
acentrosomal plant microtubule nucleation from existing
6 Cell Cycle Modules in Plants for Entry into Proliferation and for Mitosis
Fig. 6.4 CDK associated with CYCB1 is required for g-tubulin
mediated microtubule nucleation and organization of mitotic and cyto-
kinetic microtubular arrays in acentrosomal plant cells. Upper panel:
Vicia faba root meristem cells at metaphase stained for a- and g-tubulin
and for DNA with DAPI. g-Tubulin is co-localized with the kineto-
chore fibres of the microtubular spindle. g-Tubulin shows a gradient,
being most abundant in the vicinity of spindle poles (asterisk) away
from the kinetochores. Inhibition of mitotic CDKs by the drug
roscovitine results in aberrant metaphase spindles; only remnants of
kinetochore microtubular fibres persist (arrows). g-Tubulin is not
associated with these remnants of aberrant microtubule fibres, but is
dispersed within the cytoplasm, suggesting that CDK might regulate g-
tubulin association with microtubules, or g-tubulin-mediated microtu-
bule nucleation. Lower panel: Triple immunofluorescence labelling for
a- and g-tubulin and for DNA with DAPI in wild type tobacco BY-2
cells, and in cells where a non-destructible mutant CYCB1 form
is over-expressed (cycB1mut). In late anaphase, g-tubulin label
microtubules (Murata et al. 2005). AtAurora1 and the
AtTPX2 regulatory module may act with the g-tubulin
nucleation complex and other substrates involved in the
spatio-temporal regulation of acentrosomal plant spindle
formation.
91
accumulates in the region with shortening kinetochore fibres on poles
(asterisk) and forms a gradient in the midzone with enrichment at sites
adjacent to chromatin (arrows) where phragmoplast microtubules are
nucleated. At telophase, g-tubulin is localized with the phragmoplast
microtubules. Cells over-expressing a destruction box mutant form of
CYCB1 (cycB1mut) maintain an abnormally high CDK activity at
anaphase. g-Tubulin in these cells neither localizes to anaphase poles
(asterisks) nor accumulates in the midzone adjacent to separating
chromatin (arrows). We find an abnormal localization pattern for g-
tubulin during the anaphase/telophase transition and a delayed transi-
tion from the mitotic spindle organization into phragmoplast. As a
consequence, the microtubular cycle lags behind the chromatin cycle.
g-Tubulin becomes completely dispersed in cells with strong cycB1mut
expression; the phragmoplast and consequently the cell plate are absent
and cells become binuclear (empty arrow), eventually cells initiate
anaphase but lack nuclear division and cytokinesis (arrowhead), and
the next interphase is restored without regular chromatin
There is growing evidence to suggest a non-canonical role
for g-tubulin as a multifunctional protein involved in various
cellular processes, including cell cycle regulation. Indepen-
dent of microtubule assembly, g-tubulin down-regulation in
Arabidopsis compromises cytokinesis (Binarová et al. 2006).
92
CyclinB1 degradation and inactivation of mitotic kinases are
prerequisites for mitotic exit and for the completition of cyto-
kinesis (Fig. 6.4). In higher plants, microtubules are organized
without a well defined microtubule organizing centre
(MTOC). At anaphase, the acentrosomal mitotic spindle is
formed by shortening microtubular fibres on poles and the
midzone microtubules are transformed into the microtubular
cytokinetic apparatus, called the phragmoplast. It is still not
known whether phragmoplast microtubules are nucleated
de novo from dispersed sites in the midzone analogous to the
equatorial microtubule nucleation sites of Saccharomyces
pombe or whether phragmoplast is formed by capturing
preformed microtubules (J€ urgens 2005). Cytokinesis is
severely impaired if there is over-expression of nondegradable
cyclin B and mitotic activity of CDK in late mitosis (Fig. 6.4)
(Weingartner et al. 2004). Coordination between mitosis and
cytokinesis is lost in g-tubulin-conditional mutants of
S. pombe (Hendrickson et al. 2001) and g-tubulin mutants of
Aspergillus, where the function of mitotic checkpoint proteins
controlling entry into anaphase is impaired (Prigozhina et al.
2004). Failure to inactivate the anaphase promoting complex/
cyclosome in g-tubulin mutants suggests that regulation of
cell division through g-tubulin may be mediated by its inter-
action with the APC complex (Nayak et al. 2010).
The accumulating evidence demonstrating the presence
of g-tubulin in both plant and mammalian nuclei (Binárova
et al. 2000; Lesca et al. 2005) has led to the intensive search
for its nuclear function. g-Tubulin is phosphorylated by the
cell cycle inhibitory WEE1 kinase in Drosophila embryo
extracts (Stumpff et al. 2005). Also, g-tubulin was shown to
mediate the binding of hypophosphorylated Breast Cancer
Associated 1 (BRCA1) protein with the mitotic centrosome
(Hsu et al. 2001). During S and G2-phases BRCA1-
dependent ubiquitin ligase directs ubiquitination of g-tubulin
resulting in the inhibition of centrosome microtubule nucle-
ation (Parvin 2009). Microtubular and nuclear localization
of g-tubulin requires a functional Nuclear Localization Sig-
nal (NLS) sequence; activation by CDK1; association with
BRCA1 for loading of g-tubulin to microtubules; and trans-
portation of the BRCA1 g-tubulin complex to the nucleus.
All these are required for the g-tubulin function in DNA
damage response. (Hubert et al. 2011). Coupling of DNA
damage checkpoints with the centrosomal cycle is further
supported by data showing that nuclear g-tubulin interacts
with the tumour suppressor C53 protein (Hořejši et al. 2012).
The g-tubulin forms a complex with the E2F transcription
factor during the G1/S transition and the interaction of
nuclear g-tubulin with E2F was found to be important for
the G1/S phase checkpoint (Hoog et al. 2011). Moreover,
g-tubulin regulates E2F activity by competing with DP1 in
the E2F/DP1 complex. Down-regulation of g-tubulin or its
mutation causes a delay of S-phase but an increase in E2F
activity, accompanied by an enrichment of E2F regulated
transcripts. The NLS sequence of g-tubulin is essential for its
Z. Magyar et al.
interaction with the E2F proteins during G1/S regulation.
These results provide further evidence that g-tubulin is
essential for all major transitions of the cell cycle, including
G1/S, G2/M and the mitotic exit. However, the molecular
mechanisms as to how g-tubulin regulates these checkpoints
are not well understood. It could equally be that g-tubulin
acts alone or in complex with GCPs as scaffolding proteins,
or it may be part of the checkpoint sensors.
Acknowledgement We thank Dr. Věra Cenklová for providing
pictures of V. faba for Fig. 6.4.
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 Endopolyploidy in Plants
7
Jolanta Maluszynska, Bozena Kolano, and Hanna Sas-Nowosielska

Contents 7.1 Introduction

7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
7.1.1 Definition and Types of Endopolyploidy . . . . . . . . . . . . . . . . . 99 Plant growth and development is precisely programmed and
7.1.2 Polysomatic and Non-polysomatic Plants . . . . . . . . . . . . . . . . . 101
achieved through three processes: cell division (prolifera-
7.1.3 Pattern of Endoreduplication: Polysomaty . . . . . . . . . . . . . . . . 101
tion), growth and differentiation. These three processes may
7.2 Molecular Mechanisms of Endoreduplication . . . . . . . . . 102 overlap during plant organ development, when some cells
7.2.1 Endoreplication Onset: Transition from Mitotic Cell
Cycle to Endocycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
start to differentiate while others continue to divide e.g., leaf
7.2.2 DNA Replication in the Endocycle . . . . . . . . . . . . . . . . . . . . . . . 103 epidermal cells (Harashima and Schnittger 2010). Dividing
7.2.3 Endocycle Regulatory Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 cells, called meristematic cells, increase their number and
7.2.4 Factors Involved in Endopolyploidy Modulation . . . . . . . . . 106 supply new cells for post-embryonic plant development.
7.3 Structure of the Endopolyploid Plant Nucleus . . . . . . . . 108 Outside the meristems non-dividing cells expand and differ-
7.4 Occurrence of Endopolyploidy . . . . . . . . . . . . . . . . . . . . . . . . . . 108 entiate. Cell proliferation and expansion result in varied but
7.4.1 Endopolyploidy in Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 determined cell sizes specific for the plant, organ and tissue.
7.4.2 Life Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 The next phase in plant development is cell-type specifica-
7.4.3 Endopolyploidy in Generatively Polyploid Plants . . . . . . . . 109 tion along with the differentiation processes. The control of
7.4.4 Endopolyploidy in Plant Development . . . . . . . . . . . . . . . . . . . . 110
all processes and the determination of final cell mass and
7.5 Polysomatic and Non-polysomatic Plants in Culture In size are poorly understood but there is increasing knowledge
Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
about the molecular mechanisms underpinning the regu-
7.6 Methods to Analyse Endopolyploidy . . . . . . . . . . . . . . . . . . . . 114 latory systems. Cell sizes in plants are usually closely related
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 to their function. There are two strategies to enlarge cell size:
one is based on water uptake and vacuolar growth and the
other is to increase the nuclear DNA content or the level of
polyploidy, this gives rise to endopolyploidy.

7.1.1 Definition and Types of Endopolyploidy

Endopolyploidy is a general term defining the results of the

exponential multiplication of nuclear DNA (2n) in the
absence of mitosis. Endopolyploidisation is mainly the result
of either the endoreduplication or the endomitosis pathways
(D’Amato 1964). Other processes such as the fusion of
nuclei or the appearance of multinuclear cells also lead to
polyploidisation, but they are rare and not essential for plant
development (D’Amato 1984). Exceptions are the acyto-
J. Maluszynska (*)
kinetic mitotic activity in the anther tapetum of most seed
Department of Plant Anatomy and Cytology, University of Silesia,
Jagiellonska 28, 40-032 Katowice, Poland plants and in the antipodals and endosperm of some angio-
e-mail: jolanta.maluszynska@us.edu.pl sperm taxa.

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 99

DOI 10.1007/978-3-7091-1160-4_7, # Springer-Verlag Wien 2013
100
Fig. 7.1 Aconitum ranunculifolius (2n ¼ 16), highly endopolyploid
nuclei from the antipodals in the embryo sac; (a) fully developed giant
nucleus with diffuse chromatin structure (note a 2C nucleus from the
7.1.1.1 Endomitosis
Endomitosis was first described by Geitler (1939) in the insect
Gerris lateralis (Heteroptera) as the process of chromosome
division without cell division. During the endomitotic cycle
chromosomes double and condense (endoprophase, endo-
metaphase) within an intact nuclear envelope. The sister
chromatids then separate and decondense in the nucleus lead-
ing to endopolyploidy, which is characterised by the multipli-
cation of the visible chromosome number. Endomitotic cycles
of this type have been reported in several animal groups
but rarely in angiosperms (D’Amato 1989; Weiss and
Maluszynska 2001).
7.1.1.2 Endoreduplication
The most common mode of plant cell polyploidisation is
endoreduplication. This process was first described by Levan
(1939) in the elongation zone of Allium cepa roots. It is
estimated to occur in over 90% of all angiosperms and is also
present in algal and fern cells but absent in gymnosperms
(Barow and Meister 2003). Endoreduplication is a process in
which chromosomes replicate without a subsequent nucleus
and cell division and without any obvious signs of chromatin
condensation and decondensation. As a result, chromosomes
have 2n chromatids, which may become visible when such a
nucleus is stimulated to enter mitosis again. The chromatids in
interphase may fall apart or remain united at certain regions,
mostly the centromeres, or, very rarely, along most of their
length. In the latter case chromatid bundles, so-called giant
chromosomes, are formed. Many successive cycles of DNA
replication without the segregation of sister chromatids may
result in polytene chromosomes. As proposed by Huskins
(1947), the lowest level of polyteny is represented by a
J. Maluszynska et al.
integument at the bottom right hand corner of the figure); (b) nucleus
with “giant chromosomes” in haploid number (n ¼ 8). Bar: 5 mm
(From Tschermak-Woess (1956b), with permission of Springer)
diplochromosome (¼ 4 chromatid chromosome), which is the
result of two successive rounds of DNA replication. The best-
known examples of true polyteny are the giant salivary gland
chromosomes of Drosophila. The characteristic of polyteny is
the cable-architecture with banded appearance of the chromo-
some. In plants, giant chromosomes resembling polytene
chromosomes of the dipteran type have been observed in
only a few species of Phaseolus and Vigna in ovary or imma-
ture seed tissue (embryo suspensor) and the anther tapetum
respectively (Carvalheira 2000) or in the antipodal cells of
Aconitum ranunculifolius (Fig. 7.1) and specialized cells of
the embryo, embryo sac and endosperm of a few other plant
species (Tschermak-Woess 1956a, b, 1963).
While endomitosis always leads to a visible multiplication
of chromosomes, endoreduplication can have different effects,
depending on the chromatin characteristics (Fig. 7.2). Various
intermediate DNA configurations can occur, differing in the
degree of association between duplicated chromatids (Edgar
and Orr-Weaver 2001). Both processes, endomitosis and
endoreduplication, differ from mitosis by the absence of
mitotic spindle formation, and the presence of a nuclear enve-
lope during the entire endocycle.
Although endoreduplication is widespread in eukaryotes,
many questions and problems remain. The role and control
of the endocycle are especially poorly characterised in
plants. Nevertheless, there is a growing interest in this
field, particularly focused on (1) the switch between cell
proliferation and cell differentiation, and between the
mitotic cycle and the endocycle, and (2) the termination of
the endoreduplication process. Increasing knowledge about
the mechanisms of plant cell cycle regulation should con-
tribute to a better understanding of the regulation of the
7 Endopolyploidy in Plants
Fig. 7.2 Nuclei during cell cycle and endopolyploidisation. (a) Sche-
matic changes in chromosome number and DNA content, (b–d)
Gibbaeum heathii (2n ¼ 18), interphase nuclei of raphide cells of
secondary leaf; (b) 2C nucleus, (c) presumably 16C nucleus with single
chromocentres in multiplied number, (d) presumably 16C nucleus with
endochromocentres in diploid number. Bar: 5 mm (from Schlichtinger
(1956) with permission of Springer)
endocycle and the biological significance of endopolyploi-
disation in plant development. Recent genetic studies,
mostly utilising Arabidopsis thaliana mutants, have revealed
the key genes involved in plant endopolyploidy regulation,
while progress in flow and image cytometry has accelerated
investigations and determinations of the endopolyploi-
disation pattern in new plant species.
7.1.2 Polysomatic and Non-polysomatic Plants
Endopolyploidisation manifests itself at varying ploidy
levels (labelled as 2C, 4C, 8C, 16C . . ..) of different
cells in the same individual as a result of several rounds
101
of DNA synthesis without mitosis. The variation of cell
ploidy levels is also designated as somatic polyploidy.
Endopolyploidisation is closely associated with cell
differentiation and is essential for normal development
and physiology in many different organs of plants and
animals. Plants which undergo endopolyploidisation are
called polysomatic plants in contrast to non-polysomatic
plants, whose cells remain diploid throughout differentia-
tion (Fig. 7.3).
Endoreduplication is widespread in eukaryotes and is espe-
cially common in angiosperms; however, it is not present in
all species and what determines which species undergo a
polysomatic or non-polysomatic (non-endopolyploid) type
of development is unclear. For example, why some cells
undergo endopolyploidisation in one species in the early
stage of development (e.g., Chenopodium quinoa, Kolano
et al. 2009) while in others all cells remain diploid and their
non-dividing nuclei stay at the 2C DNA level (e.g., Crepis
capillaris, Brossard 1978; Maluszynska 1990) is poorly
understood. Non-polysomatic plants seem to be typical for
some plant families. The dominance of non-polysomatic spe-
cies has been documented in such families as Amaryllidaceae,
Asteraceae, Fagaceae, Liliaceae, Ranunculaceae, Rosaceae
and others (Barow and Jovtchev 2007).
7.1.3 Pattern of Endoreduplication:
Polysomaty
Endopolyploidy in polysomatic plants can be characterised
in two ways: (1) the frequency of endopolyploid cells and (2)
the degree of ploidy in the particular cells of tissues and
organs. These two factors determine the pattern of endopoly-
ploidy, which is characteristic for each species and is known
to have an organ-specific character. The degree of endopoly-
ploidy is indicated by the C level where 1C is the DNA
content in unreplicated gametophyte nuclei and gametes. 2C
and 4C represent the DNA amounts of mitotically active
sporophytic cells in the G1 and G2 phase of the cell cycle,
respectively. Cells with a DNA content higher than 4C are
considered to be endopolyploid. Generally, during endo-
polyploidisation nuclei undergo several rounds of endoredu-
plication, reaching levels of 64C DNA. It should be noted
that the 4C population may comprise not only of cells in the
G2 phase of the cell cycle but also differentiated cells that
have already gone through the first round of endoredu-
plication. However, in mature organs mitotically active
cells are not present or their frequency is very low. Higher
levels of endoreduplication occur in metabolically highly
active cells that have a secretory or nutritive function. Mul-
tiple cycles of endoreduplication have been described in
many specific cell types like antipodal cells, suspensor
cells, endosperm haustoria or tapetum cells (Table 7.1). An
102 J. Maluszynska et al.

Table 7.1 Examples of endopolyploidy in specific plant tissues potential substrates kinase will interact with. Two cyclin
(Mauseth 2009) families seem to be involved in the endocycle: CYCD and
Species Tissue Degree CYCA. CYCD drives the cells into S-phase, while CYCA
Carex hirta Tapetum 8C probably controls the progression of S-phase (Joubes and
Viola declinata Elaiosome 16C Chevalier 2000).
Cucumis sativus Anther hairs 64C
Triticum aestivum Antipodal cells 128C
Geranium phaeum Integument 512C 7.2.1 Endoreplication Onset: Transition from
Phaseolus vulgaris Suspensor 2,048C Mitotic Cell Cycle to Endocycle
Scilla bifolia Elaiosome 4,096C
Phaseolus coccineus Suspensor 8,192C
The transition from the mitotic cycle to the endocycle is
Arum maculatum Endosperm 24,576C
irreversible and controlled by genetic and environmental
cues. The process is regulated by several pathways which
are thought to lead to the inactivation of the mitosis-
extremely high DNA content, up to 24,576C, which promoting factor (MPF) and provide the oscillatory activity
corresponds to 13 endocycles, has been reported for the of S-phase CYC-CDK complexes. MPF inactivation
endosperm cells of Arum maculatum (Traas et al. 1998). prevents the cells from passing the G2/M transition check-
point, while the oscillatory CDK activity enables DNA
re-replication. Low kinase activity is thought to allow pre-
replication complexes (pre-RC) to set up at the origin of
7.2 Molecular Mechanisms replication (ORI) in the chromatin, while high kinase activ-
of Endoreduplication ity permits the replication machinery to proceed and copy
the DNA (Sugimoto-Shirasu and Roberts 2003). This
During the mitotic cell cycle, cells undergo sequential oscillatory activity pattern is achieved mostly by utilising
phases of DNA replication (S-phase) and chromosome common cell cycle machinery with only a few proteins
segregation (mitosis—M-phase), allowing chromosome rep- found to be specific for endoreduplication (Dewitte et al.
lication only once per cell cycle. Replication is preceded by 2007; Ishida et al. 2008; John and Qi 2008; Chevalier et al.
the gap phase (G1) during which cells grow and synthesise 2011) (Fig. 7.4a).
the proteins needed for S-phase. A second gap phase (G2) Little is known about how MPF inactivation is achieved;
also separates the S-phase and mitosis. During the gaps however, studies of Arabidopsis have shed some light on this
several checkpoints operate which regulate cell cycle pro- topic. The family of CDKB1s has been shown to drive the
gression. The checkpoints act in response to intra- and extra- cells through the G2/M transition during the cell cycle
cellular regulatory signals (Fig. 7.4a). (Menges et al. 2006; Gutierrez 2009). Within this family,
A particular class of protein kinases, known as CYCLIN CDKB1;1 seems to play a special role and its high activity is
DEPENDENT KINASES (CDK) along with their regulatory crucial for keeping the cells in the cell cycle and preventing
proteins, known as CYCLINS (CYCs), are involved in the them from entering the endocycle (Boudolf et al. 2004,
progression through the cell cycle. Two main families of 2009). Indeed, analysis of Arabidopsis transformants with
CDKs act during the cell cycle: (1) continuously active reduced CDKB1;1 activity has shown that the down-
CDKA and (2) temporarily activated, mitotic CDKBs regulation of CDKB1;1 results in the onset of the endocycle
(Fig. 7.4a). These CDKs form complexes with a variety of (Boudolf et al. 2004). This downregulation in wild type
cyclin family members. Cyclic changes in cyclin levels plants is probably achieved by a decrease in the availability
result in changes in the activity of the CYC-CDK of the cyclin CYCA2;3 through its selective degradation, as
complexes, thus allowing phase succession (Dewitte and the stability and activity of CDKB1;1 is dependent on its
Murray 2003; see also Magyar et al. 2013, this volume). interactions with CYCA2;3 during the G2/M transition. It
When cells start to differentiate they stop dividing and has also been suggested that the CYCA2;3-CDKB1 complex
may enter endoreduplication which operates through a is responsible for the temporal downregulation of the CYC-
modified cell cycle called an endocycle. A single endocycle CDKA1 activity, which might result in blockage of the
consists of just the G1 and S-phase, i.e., it lacks the G2 and G2/M transition (Verkest et al. 2005a; Menges et al. 2005;
M-phases (Edgar and Orr-Weaver 2001). Based on available Gutierrez 2009).
data, the entire endocycle operates through the action of Precise data about how the cells are able to undergo
CDKA1 (Leiva-Neto et al. 2004). Successive rounds of multiple rounds of DNA replication are lacking; however,
replication separated by G-phases are executed by changes it seems that this involves changing the activity of members
in a CDKA1 cyclin partner. Cyclin determines which of the of the CYCD family. Among CYCD family members,
7 Endopolyploidy in Plants
160
120
counts
80
40
0
1 10
b
320
240
counts
160
80
0
1 10
Fig. 7.3 Flow histograms of relative fluorescence of nuclei isolated
from hypocotyls from (a) polysomatic (Brassica campestris) and
(b) non-polysomatic (Helianthus annuus) plants. In (a) four peaks are
visible showing endopolyploidy up to 16C DNA while in (b) the large
CYCD3;1 has been shown to be necessary for keeping cells
in the cell cycle. Studies of Arabidopsis mutants have shown
that CYCD3;1 acting in response to a cytokinin signal is
involved in the maintenance of the cell cycle while its
downregulation seems to be needed for the onset of
endoreduplication (Dewitte et al. 2007).
7.2.2 DNA Replication in the Endocycle
The G1/S transition and DNA replication are mainly
regulated by the S-phase specific cyclin family CYCD
(Menges et al. 2006). During the endocycle a sub-family of
CYCD1 seems to play an important role. It has been shown
that one of the main targets of the CYCD1-CDKA1 complex
is RETINOBLASTOMA-RELATED protein (RBR), which
forms an inhibitory complex with the E2FA transcription
103
4C
8C
2C
16C
100
FL4 - DAPI
2C
4C
100
FL4 - DAPI
peak indicates that most nuclei are 2C. The second, much smaller peak
shows that there are, however, a few nuclei with a 4C DNA amount.
x-axis nuclear log DAPI fluorescence intensity, y-axis number of nuclei
factor (E2FA TF) when it is unphosphorylated (Joubes and
Chevalier 2000; Park et al. 2005; Gutierrez 2009). The
phosphorylation of RBR, via the action of the CYCD1-
CDKA1 complex, releases the E2FA TF allowing it to
form a complex with the DIMERISATION PARTNER A
(DPA) protein. The E2FA-DPA complex then induces the
expression of the main replication proteins. Consequently,
the action of the E2FA-DPA allows the pre-RC to assemble
and licences the start of the next S-phase (Joubes and Che-
valier 2000; del Castellano et al. 2001, 2004; Park et al.
2005; Berckmans and De Veylder 2009).
DNA replication during the endocycle also involves the
specific action of various proteins not found during the
mitotic cell cycle replication. Topoisomerase VI (TOPOVI)
is a plant-specific topoisomerase involved in chromosome
decatenation. Analysis of Arabidopsis mutants with non-
functional components of TOPOVI complex has shown
that they only undergo the first two rounds of
104 J. Maluszynska et al.

70,0
65,0
60,0
55,0
50,0
45,0
40,0
Counts

35,0
30,0
25,0
20,0
15,0
10,0
5,0
0,0
0,0 2000000,0 5000000,0
Total Intensity DAPI

Fig. 7.4 (a) Schematic representation of the cell cycle (left) and the
endocycle (right). The cell cycle consists of two main phases: replica-
tion (S-phase) and division (M-phase), separated by G1 and G2 phases.
Transition from one phase to another is determined by the cyclin-
dependent kinases (CDK)—CDKA and CDKB. CDKA is present con-
stantly during the cell cycle, while CDKB is considered to be specific
for the G2/M transition. CDK activity and substrate specificity are
regulated by cyclin (CYC) proteins, thus the periodic presence of
different CYCs, determines cell cycle progression. The anaphase pro-
moting complex (APC) participates in the degradation of mitotic
cyclins, thus preventing cells from entering mitosis prematurely. The
switch from the cell cycle to the endocycle involves a decrease in
the activity of CYC-CDKB complexes and inactivation of CYCA2;3.
The endocycle is considered to be the cell cycle without the M-phase,
thus it is regulated by similar mechanisms. As for the cell cycle, its
progression is regulated by the oscillatory activity of CYCs, however
only CYCA and CYCD have been shown to be active during the
endocycle. Constant activity of APC ensures that nuclei do not enter
mitosis. (b) Changes in the nuclear morphology and chromocenter
structure during consecutive rounds of endoreplication. Arabidopsis
thaliana stem leaf nuclei after DAPI staining (left) and fluorescence
in situ hybridization with the centromeric probe, pAl1 (right). In nuclei
with low ploidy levels centromeric heterochromatin is compacted,
indicating sister chromatid association, while in nuclei of higher ploidy
levels centromeric heterochromatin is decondesed and appears diffuse.
(c) Analysis of the DNA content in A. thaliana stem leaf nuclei using
image cytometry. Slides with stem leaf nuclei were stained with DAPI
and examined under the microscope. The DNA contents of the nuclei
were evaluated by measuring fluorescence intensity (x-axis) using
image cytometry and the results are displayed as a histogram. Peaks
represent consecutive ploidy levels (2C, 4C and >4C), determined
from relative fluorescence intensity. Pictures of representative nuclei
for each ploidy level are shown over each peak. Bar: 5 mm
7 Endopolyploidy in Plants
endoreduplication and fail to succeed in the following
rounds, suggesting a role of TOPOVI in proper DNA repli-
cation in nuclei with higher ploidy levels (Sugimoto-Shirasu
et al. 2002; Kirik et al. 2007; Sugimoto-Shirasu et al. 2005).
There is also evidence for the involvement of the chro-
matin assembly factor 1 (CAF-1) in the regulation of the
endocycle. CAF-1 is involved in the assembly of nucleo-
somes onto a newly synthesised DNA strand. In caf-1
mutants, nuclei were seen to go through one additional
endocycle on average. It therefore seems that the replication
machinery is also involved in the regulation of the number of
endocycles (Exner and Henning 2008; Gutierrez 2009).
7.2.3 Endocycle Regulatory Proteins
7.2.3.1 Transcription Factors in the Endocycle
Control of the endocycle is provided at various levels by
many pathways that are interdependent and often form feed-
back loops (Edgar and Orr-Weaver 2001; Lee et al. 2009). It
has been shown that the basic steps needed for DNA repli-
cation remain under the transcriptional control of the E2F TF
family. These factors can be divided into typical and atypical
TFs, depending on whether their action requires dime-
risation with DP proteins or whether they work as
monomers, respectively. In Arabidopsis typically E2Fs act
either as transcriptional activators (E2FA and E2FB) or
repressors (E2FC) and their main action takes place during
the G1/S transition (del Castellano et al. 2001, 2004; Sozzani
et al. 2006). E2F TFs may also be involved in the onset of an
endocycle, as their target elements have been found in the
promoter region of CDKB1 and CDKB1;1 (Boudolf et al.
2004; del Pozo et al. 2006). The group of atypical E2F TFs
work as transcriptional repressors (Berckmans and de
Veylder 2009). Several studies have provided evidence for
their role in the regulation of the transition from the cell
cycle to the endocycle, e.g., the DP/E2F-like protein DEL1
has been found to control entry into endoreduplication,
operating upstream of the proteosomal degradation pathway
(Vlieghe et al. 2005; Lammens et al. 2008). Apart from E2F
TFs, whose actions are the best studied, other transcription
factors have been shown to impact both the onset of an
endocycle and their number. Most of them are involved in
CYC and CDK gene transcription; however, their precise
mode of action is unknown (Wang et al. 2006; Berckmans
and De Vevlder 2009; Tojo et al. 2009).
7.2.3.2 Protein-Protein Interactions in the
Regulation of an Endocycle
During endoreduplication an important role is played by the
posttranslational control of various regulatory proteins.
These proteins can be divided into two groups, depending
105
on whether they execute their action directly, binding to their
target, or indirectly, by chemically modifying their target.
The first type of action is performed by the INHIBITOR/
INTERACTOR OF THE CDK/KIP-RELATED PROTEINS
(ICK/KRP), the plant CYCLIN DEPENDENT KINASE
INHIBITORS (CKIs). ICK/KRPs most probably execute
their inhibitory action by binding to CYC-CDK complexes
and inhibiting their kinase activity (Verkest et al. 2005a).
Particular members of ICK/KRP show substrate specificity,
which is determined by the cyclin involved in the CYC-CDK
complex (Verkest et al. 2005a; Wang et al. 2006). However,
even though their main action seems to be focused on CYC-
CDK complexes, ICK/KRPs can also interact with cyclins
(Coelho et al. 2005; Wang et al. 2006). Several studies have
highlighted the role of ICK/KRPs in the onset and progres-
sion of an endocycle in a concentration-dependent manner
(Schnittger et al. 2003; Coelho et al. 2005; Verkest et al.
2005b; Bisbis et al. 2006). One of the best studied ICK/KRP
members is Arabidopsis SIAMESE (SIM), a crucial regula-
tor of the onset of an endocycle in trichomes (Churchman
et al. 2006). Mutants with truncated SIM protein produce
multicellular trichomes with nuclei possessing lower ploidy
levels, compared with wild-type plants. Experiments using
Fluorescence Resonance Energy Transfer (FRET) methods
have shown that SIM interacts with CYCD and CDKA1,
blocking the G2/M phase transition and allowing the onset of
an endocycle (Churchman et al. 2006; Kasili et al. 2010).
Depending on the effect of their action, proteins
introducing chemical modifications can be divided into
inhibitors or activators. Among the inhibitors, is WEE1
kinase which has been shown to play an important role during
the cell cycle and is also involved in the endocycle. WEE1 is
involved in M-phase inhibition, probably acting through
CDKA1 phosphorylation. Tomato transformants with an
impaired expression of Solly:WEE1 were generally smaller
and developed smaller fruits with lower ploidy levels com-
pared with wild-type plants. (Sorrell et al. 2002; Gonzalez
et al. 2007). Activating phosphorylation is performed by
CYCLIN-DEPENDENT KINASE ACTIVATING KINASE
(CAK). It has been suggested that one of the CAKs
(CDKF;1), which is necessary for activation of the CYC-
CDKA complex during the cell cycle, plays a crucial role
as a positive endocycle regulator (Umeda et al. 2000;
Shimotohno et al. 2006; Takatsuka et al. 2009).
It has also been shown that SUMO (small ubiquitin-like
modifier) ligase E3 may act as an endocycle repressor in
different cell types. Seedlings of Arabidopsis mutants with
inactive SUMO ligase only live up to three weeks and
show severe developmental defects. These defects concern
the cell cycle, and result in the malformation of the meri-
stem as well as extra rounds of endoreplication in leaf cells
(Ishida et al. 2009). The precise mechanism in which
SUMOylation affects an endocycle still needs to be
106
evaluated; however, cyclin, CDK as well as the E2F genes
seem to be potential targets. A possible SUMO action over
the structure maintenance of chromosome (SMC) complex
has also been proposed (Ishida et al. 2009).
7.2.3.3 Protein Degradation
The molecular pathways driving the protein degradation
necessary for an endocycle remain largely unsolved. It is
generally known that endocycle protein activity is controlled
through a ubiquitination-dependent proteasomal-degradation
pathway. Three enzymes are responsible for performing
ubiquitination: ubiquitin activating enzyme (E1), ubiquitin
conjugating enzyme (E2), and ubiquitin ligase (E3). The
E3 enzyme is a multisubunit complex that performs
ubiquitination and is responsible for its substrate specificity
(Vierstra 2009). During the endocycle, as in the cell cycle,
two E3 ligase types have been shown to be involved:
ANAPHASE-PROMOTING COMPLEX (APC) and SKP,
CULLIN, F-BOX CONTAINING COMPLEX (SCF)
(Kondorosi and Kondorosi 2004; Hershko 2005). Both
APC and SCF are nuclear proteins which direct proteins
destined for degradation into the 26S proteasome pathway.
The specificity of their action is determined by several
regulatory subunits which are different for each complex
(Vierstra 2009). It has been suggested that APC action
focuses on regulating the availability of cyclin, while SCF
targets different regulatory proteins as well as the E2F tran-
scription factors (Hershko 2005; Del Pozo et al. 2006; Ren
et al. 2008). In different organs the APC substrate specificity
is regulated by different factors. For example in Arabidopsis,
CCS52A and CCS52B proteins determine the substrate
specificity of the APC (Kondorosi and Kondorosi 2004;
Li et al. 2009). The CCS52A2 regulatory subunit is respon-
sible for driving the ubiquitin-dependent degradation of
CYCA2;3 in leaves, whereas a similar role is played by
CCS52A1 in roots (Lammens et al. 2008; Boudolf et al.
2009). The role of proteasomal degradation has also been
shown for other proteins such as members of pre-RC: CDC6
or CDT1 (del Castellano et al. 2001, 2004). Generally, the
constant activity of both APC and SCF, which change their
substrate specificity during the endocycle, seems to provide
the mechanism by which the oscillatory activity of CDK is
achieved. Recently Sako et al. (2010) showed that a muta-
tion in one of the subunits of the 26S proteasome regulatory
particle affected the number of endocycles in Arabidopsis
trichomes, thus indicating the direct involvement of the 26S
proteasome pathway in the regulation of an endocycle.
7.2.3.4 Determination of the Number
of Endocycles
Each cell type has a determined ploidy level that indicates
that the DNA replication cycles need to be stopped at a
J. Maluszynska et al.
particular time (Joubes and Chevalier 2000; Jovtchev et al.
2007; Lee et al. 2009). Current data point to several proteins
that seem to be specifically involved in the exit from the
endocycle. Imai et al. (2006) studied Arabidopsis mutants
with a truncated action of CYCA2;3. They showed that the
loss of CYCA2;3 activity resulted in large phenotypic
changes in plants. Compared with wild-type plants the
mutants had trichomes with bigger nuclei; their roots,
cotyledons, and first leaves had cells with higher ploidy
levels; and the first endocycle occurred earlier in develop-
ment. Molecular studies have shown that the CYCA2;3
activity is negatively correlated with cell ploidy levels and
that CYCA2;3 is able to form an active complex with
CDKA1 in plants. Thus, it has been suggested that the
CYCA2;3-CDKA1 complex is involved in the termination
of an endocycle, probably by the down-activation of the pre-
RC complexes. Moreover, studies of Arabidopsis trichomes
have also pointed to the role of GT2 LIKE1 (GTL1) in the
termination of an endocycle. GTL1 belongs to the trihelix
TF family and acts downstream of TRANSPARENT
TESTA GLABRA (TTG1) and GLABROUS 2 (GL2) TFs
(Breuer et al. 2009). Unfortunately, the precise targets of
GTL1 have not yet been identified.
7.2.4 Factors Involved in Endopolyploidy
Modulation
Endoreduplication remains under the developmental control
of various phytohormones. The best studied are the actions
of gibberellin (GA), auxin and cytokinins (see below); nev-
ertheless, other phytohormones are also known to have an
impact on the endocycle. Ethylene has been shown to be
involved in regulating the number of endocycles in
Arabidopsis hypocotyls. Treating wild-type plants with an
ethylene precursor resulted in extra rounds of endorep-
lication (Gendreau et al. 1999). The actions of brassi-
nosteroids seem to be involved in maintaining cells in the
endocycle. Studies on the Arabidopsis brassinosteroid-
insensitive mutant bin4-1 showed that leaf ploidy levels
did not exceed 8C, whereas in wild-type plants they reached
32C. This mutation resulted in the truncation of the TOPOVI
complex and was correlated with an increased level of
CDKB1;1, an endocycle inhibitor (Breuer et al. 2007). In
contrast, abscisic acid (ABA) has been shown to have a
negative impact on the progress of an endocycle. Working
during the G1/S transition, ABA is probably involved in the
regulation of genes involved in replication and CDK regula-
tion (del Castellano et al. 2004; del Pozo et al. 2005; Wang
et al. 2006). It has also been shown that environmental
factors including light and nutrient availability affect an
endocycle and are involved in determining their number.
7 Endopolyploidy in Plants
7.2.4.1 Gibberellic Acid
Gibberellic Acid (GA) signalling is known to be involved in
the onset of an endocycle during Arabidopsis thaliana tri-
chome differentiation. Using a wide variety of A. thaliana
trichome mutants with disrupted GA signalling, many
proteins participating in the GA signalling pathway have
been discovered. It has been shown that during trichome
differentiation GA acts in a concentration-dependent manner
activating various transcription factors (Perazza et al. 1998;
Ishida et al. 2008). Among the genes directly activated by
those regulators are the key genes involved in the onset and
progress of an endocycle like: RBR1, SIM and ICK1/KRP1.
During trichome differentiation RBR1 is probably involved
in the determination of the number of endocycles, whereas
SIM participates in the induction of endocycles (Churchman
et al. 2006; Morohashi and Grotewold 2009). Along with
SIM, ICK1/KRP1 also plays a role in the induction of
endocycles in trichomes by downregulating the activity of
mitotic CYC-CDK complexes (Schnittger et al. 2003).
7.2.4.2 Auxin
In the body of a plant auxin forms concentration gradients
which give rise to differences in the local auxin concentra-
tion. These differences are responsible for the local character
of auxin action, including the regulation of endoredu-
plication (del Pozo et al. 2005; Gutierrez 2009). A positive
correlation between auxin concentration and the onset of an
endocycle has been shown in maize endosperm cells (Lur
and Setter 1993). On the other hand, in the roots of
Arabidopsis a low auxin concentration seems to promote
endoreduplication, whereas high concentrations determine
meristematic cell activity in the proximal meristem (Ishida
et al. 2010). Treating Arabidopsis seedlings with auxin
antagonists results in a reduction of the root meristem size.
The same results have been obtained in Arabidopsis auxin
resistant mutant 3 (axr3), which is auxin insensitive. Thus,
in the Arabidopsis root meristem, cells enter an endocycle
prematurely when auxin signalling is inhibited. Ishida et al.
(2010) suggested that high endogenous levels of auxin,
known to be present in the root division zone, have an
inhibitory effect on the onset of an endocycle in Arabidopsis
proximal root cells. Whereas in cells outside the meriste-
matic zone, where auxin concentrations are lower, the
repression of endoreduplication might be lifted allowing
cells to switch to the endocycle.
In an endocycle, auxin has been shown to act as a KRP2-
specific repressor and to modulate the activity of the E2F
transcription factors, which are known to regulate the
expression of the endocycle genes, including genes neces-
sary for DNA replication (Magyar et al. 2005; Verkest et al.
2005a; Sozzani et al. 2006). Generally, auxin can influence
107
DNA synthesis and epigenetic modifications as well as the
degradation of different proteins involved in both the cell
cycle and endocycle, and thus execute the developmental
program of the cell (Horvath et al. 2006; Ishida et al. 2009,
2010; Perrot-Rechenmann 2010).
7.2.4.3 Cytokinins
Cytokinins have been shown to regulate the transcription of
the main cell cycle genes, and thus are considered to be one
of the main factors affecting the proliferative activity of
plant cells. In most studies their action seems to be antago-
nistic to auxin (del Pozo et al. 2005). Ishida et al. (2010)
studied how the application of exogenous cytokinins
influenced the timing of the onset of an endocycle during
the differentiation of Arabidopsis root cells. After treatment
with exogenous cytokinins, an increase in the amount of
endogenous cytokinins was noticed. Studied plants also
showed a decrease in root meristem size. This led to a
suggestion that elevated cytokinin levels are responsible
for the earlier onset of an endocycle in Arabidopsis proximal
root cells. A similar analysis conducted on mutants with
disrupted cytokinin synthesis showed a significant delay in
the onset of the endocycle. However, this does not seem to
be a general rule. During the development of Arabidopsis
leaf and maize endosperm, high cytokinin levels were posi-
tively correlated with a high mitotic activity of the cells
(Lur and Setter 1993; Xia et al. 2009). The action of
cytokinins is executed by activating the dephosphorylation
of mitotic CYC-CDK complexes along with the positive
regulation of CYCD, particularly CYCD3, gene transcrip-
tion, thus keeping cells in the cell cycle. It is possible that
cytokinins might also act through directing specific proteins
to a proteasomal pathway (Riou-Khamlichi et al. 1999;
Dewitte and Murray 2003).
7.2.4.4 Light
Studies of phyA and phyB mutants of A. thaliana have provided
evidence for the involvement of the phytochromes PHYA (far
red light receptor) and PHYB (white light and red light recep-
tor), in the regulation of endoreduplication. Both phytochromes
act by inhibiting specific rounds of DNA synthesis. In the
hypocotyls of Arabidopsis seedlings grown in the light, cells
went through two rounds of replication reaching 8C ploidy
level, while in seedlings grown in the dark an extra round of
replication was observed creating nuclei with 16C (Gendreau
et al. 1998). The number of endocycles also remains under blue
light control, probably executed through the ubiquitination
pathway (Tsumoto et al. 2006). In Arabidopsis cotyledons,
the action of light stimulated the expression of the main cell
cycle genes, including those with an impact on endoredu-
plication (López-Juez et al. 2008).
108
7.2.4.5 Nutrients
Sucrose is known to regulate G1/S progression through
protein phosphatase-dependent CYCD gene induction
(Riou-Khamlichi et al. 1999; Hirano et al. 2008). Acting
down this pathway, sucrose might also have an impact on
the progression of an endocycle. Moreover, it has been
shown that nutrient starvation affecting plant growth can
also influence endoreduplication (Henriques et al. 2010).
7.3 Structure of the Endopolyploid Plant
Nucleus
The structure of a plant nucleus and the organization of
chromosomes inside, especially endopolyploid nuclei, is
still poorly understood. One of the most widely studied are
the nuclei of Arabidopsis thaliana, a polysomatic plant. The
advantage of studying these nuclei is that they possess
heterochromatic chromocenters. In meristematic cells,
chromocenters are clearly visible after fluorescent staining
with DAPI and they correspond to chromosome number.
The number of chromocenters also allows the ploidy level
of the cell to be estimated. Compared with meristematic
nuclei, endoreduplicated nuclei are bigger, in proportion to
the increase in DNA ploidy, and usually they have bigger
chromocenters (Maluszynska and Heslop-Harrison 1991;
Schubert et al. 2006; Henriques et al. 2010). However,
the increase in size is not linearly proportional to the DNA
content indicating that chromatin reorganization has occu-
rred. There is also evidence for the differential behaviour of
particular chromatin domains. For example, there is a greater
tendency for NORs (Nucleolar Organizing Regions) to fuse
in A. thaliana leaf nuclei, compared with the centromeric
regions of different chromosomes which stay preferentially
separated. However, it should be noted that NOR fusion is
most probably connected with their function in the
organization of the nucleolus (Pecinka et al. 2004; Schubert
et al. 2006; Berr and Schubert 2007).
Very little is known (with exceptions, see Sect. 7.1.1 and
Figs. 7.1 and 7.2) about the structure of endoreduplicated
chromosomes since they rarely condense and become visi-
ble. During endopolyploidisation, consecutive rounds of
replication result in the formation of chromosomes built of
numerous chromatin strands, sister chromatids, whose num-
ber increases exponentially with each endocycle. This
implies that only indirect studies of the endoreduplicated
chromosome structure and organization are possible. One
of the most useful techniques is fluorescence in situ
hybridization (FISH) using chromosome or chromosome-
region specific probes. By analysing the size and localization
of the signal insights into the structure of endoreduplicated
chromosomes can be gained (Fig. 7.4b). Indeed, using this
J. Maluszynska et al.
approach for A. thaliana leaf nuclei, it was shown that sister
chromatids stayed associated along their entire chromosome
length in nuclei with low endopolyploidy levels. However,
as ploidy level increased, separation of sister chromatids
took place (Schubert et al. 2006). Separation started at
the terminal regions, and, as the number of DNA strands
increased, this extended into the interstitial regions. With
ploidy levels over 16C, dissociation of centromeric regions
was observed (Schubert et al. 2006, 2008). It has been
suggested that sister chromatid dissociation is probably the
result of a decrease in the number of cohesin sites along the
chromosome arms (Schubert et al. 2009) and results
obtained for endoreduplicated nuclei in the endosperm of
Zea mays support this thesis. A tighter association of DNA at
centromeric and knob regions than in other chromosome
regions was also revealed by FISH (Bauer and Birchler
2006).
The chromatin structure remains under epigenetic control.
Unfortunately, very little is known about the epigenetic
changes associated with endoreduplication. Data from
Triticum durum have suggested that there is increased meth-
ylation in endoreplicated nuclei (Polizzi et al. 1998) and
recent studies on tomato fruit indicate that such modifications
may be tissue-specific (Teyssier et al. 2008). On the other
hand, a comparison of the pattern of epigenetic modifications
between A. thaliana meristematic and endoreplicated root
and leaf nuclei revealed general similarities despite an
increased acetylation of the chromatin in the chromocenters
(Jasencakova et al. 2003).
7.4 Occurrence of Endopolyploidy
7.4.1 Endopolyploidy in Species
A relationship between taxonomic position and endopoly-
ploidisation was first reported by Tschermak-Woess (1956a)
and has since been confirmed by several authors (D’Amato
1964; Nagl 1976; Olszewska and Osiecka 1982; Barow and
Meister 2003). The occurrence of endopolyploidy and the
degree of endopolyploidisation also seems to be characteris-
tic for plant families. Endopolyploidy has mainly been noted
to be common in species with a small genome size (e.g.,
Arabidopsis thaliana (Galbraith et al. 1991), Solanum
lycopersicum (Smulders et al. 1994), Lupinus cosentinii
(Hajdera et al. 2003), Brassica oleracea (Kudo and Kimura
2001a) and it has been suggested that this may be because
certain specialized cells need a minimum amount of nuclear
DNA to maintain their specific functional status, something
that can be achieved through endopolyploidisation in vege-
tative cells (Nagl 1976; Galbraith et al. 1991).
To investigate the impact of genome size and taxonomic
position on endopolyploidisation, comparative analyses were
7 Endopolyploidy in Plants
made between plants belonging to 16 phylogenetically-
diverse families covering a wide range of genome sizes in
each family (Barow and Meister 2003). Interestingly, a neg-
ative correlation between endopolyploidisation and genome
size was confirmed for only three of the investigated families
(Brassicaceae, Urticaceae and Solanaceae). Some species
with small genomes showed low levels of endopolyploidy
(e.g., Haplopappus gracilis, Aquilegia vulgaris) or were non-
polysomatic (e.g., Oryza sativa) whereas some species with
large genomes in Fabaceae or Alliaceae were polysomatic.
The authors concluded that there was only a weak but
significant negative correlation between genome size and
endopolyploidisation.
Families may differ significantly in the presence and
pattern of endopolyploidy. For instance in some families
polysomatic species with high level of endopolyploidy pre-
dominate (e.g., Amaranthaceae, Cucurbitaceae, Cheno-
podiaceae and Brassicaceae), while Solanaceae, Alliaceae,
and Fabaceae show an intermediate level and Poaceae a low
level of endopolyploidy (Barow and Meister 2003; Barow
and Jovtchev 2007). Usually, the pattern of endopolyploidy
in related species is similar but there are some exceptions
(e.g., the polysomatic species Aquilegia vulgaris belongs to
Ranunculaceae, a family dominated by non-polysomatic
species; Barow and Meister 2003).
The endopolyploidy pattern may differ between
individuals of the same species belonging to different
ecotypes or varieties. For example, the frequency of endo-
polyploid cells in roots was observed to differ between 18
ecotypes of Arabidopsis thaliana (Beemster et al. 2002).
Significantly different patterns of endoreduplication have
also been described in the endosperm of several inbred
lines of Zea mays. Flow cytometric analysis showed that
the DNA content of nuclei ranged from 3C to 48C in some
lines while in others from 3C to 192C (Larkins et al. 2001).
Similarly, different endoreduplication patterns and number
of endocycles have been reported in mature cotyledons of
different genotypes of Pisum sativum (Lemontey et al. 2000).
7.4.2 Life Strategy
The level of endopolyploidisation seems to be correlated not
only with genome size but also with the type of life cycle.
Endopolyploids are more frequent in annual rather than
perennial herbs. Among the 34 polysomatic species inve-
stigated by Barow and Meister (2003), nine were perennial
and 25 annual or biennial. Woody plants were non-
polysomatic (Barow and Meister 2003).
Analysis of endoreduplication in root cells during differ-
entiation within different monocotyledonous families
revealed a higher level of endopolyploidy in species with a
lower DNA content as well as an annual type of life cycle
109
(Olszewska and Osiecka 1982). These observations were
confirmed for the root cells of dicotyledonous herbaceous
species (Olszewska and Osiecka 1983). In general, a short
life cycle is usually correlated with a small genome size and
a high level of endopolyploidy. A good example of such a
correlation is Arabidopsis thaliana, which has a very small
genome size (157 Mb, Bennett et al. 2003), a short life
cycle and endopolyploidisation in all vegetative organs
(Galbraith et al. 1991)
7.4.3 Endopolyploidy in Generatively
Polyploid Plants
Considering the weak but significant negative correlation
between genome size and endopolyploidy, the question has
been raised as to whether polysomaty is affected by a plant’s
polyploidy level. A comparison of endopolyploidy patterns in
diploid and polyploid lines of the same species should answer
this question to see whether a certain number of endocycles is
typical for a species regardless of polyploidy level (i.e., the
number of endocycles is the same between diploid and poly-
ploidy cytotypes) or whether there is a fixed maximum DNA
amount for a species in which case the number of endocycles
is expected to differ between diploid and polyploid cytotypes.
Actually, both patterns have been observed.
Tetraploid plants of Portulaca grandiflora induced by
colchicine treatment, showed the same number of endo-
cycles as diploid plants following flow cytometric analysis
(Mishiba and Mii. 2000) and similar results have also been
obtained for artificially-induced tetraploid lines of Zea mays
(Biradar et al. 1993) and Solanum lycopersicum (Smulders
et al. 1994).
In contrast, flow cytometry analyses of diploid and
induced autotetraploid Arabidopsis thaliana plants (ecotype
Columbia) revealed different degrees of endoreduplication.
A histogram for young leaves of diploid plants showed four
peaks corresponding to a 2C, 4C, 8C and 16C DNA content
(i.e., three endocycles took place) whereas for the tetraploid
plants three peaks were observed (2C, 4C, 8C) indicating
just two endocycles had occurred (Fras et al. 2007). The
maximum DNA content was similar in diploid and tetraploid
plants but a different number of endocycles had occurred.
However, the situation for naturally occurring polyploids
may be different from artificially induced ones as no
differences in the number of endoreduplication rounds
were observed in the hypocotyls between diploid ecotypes
of Arabidopsis and the natural tetraploid Cape Verde islands
ecotype. Two endocycles occurred in both cytotypes even
though the cells of the tetraploid contained twice as much
DNA as the wild-type plant (Gendreau et al. 1998). The
results indicate that the endoreduplication pattern for natural
and artificial polyploid plants may be different. On the basis
110
of flow cytometric investigations of diploid and tetraploid
accessions and species, it was concluded that natural
polyploids showed a lower endopolyploidisation level than
the corresponding diploid suggesting that the endopoly-
ploidy level needs several generations to become established
(Jovtchev et al. 2007). This hypothesis needs more detailed
investigations and confirmation for different plant species.
7.4.4 Endopolyploidy in Plant Development
Plant development is the result of genetic predisposition and
a range of environmental parameters. It is the effect of two
processes—(1) cell division and (2) expansion which can
result from endopolyploidisation. In an individual plant
different organs can exhibit different patterns of endopoly-
ploidisation. In some species extensive endopolyploidy has
been reported in nearly all organs, while in other species it
occurs to a low degree or not in all organs and is correlated
with plant development.
7.4.4.1 Embryogenesis
Usually, the embryos of the dry fully maturated seeds of
various plant species show large amounts of cells at the
presynthetic G1 phase of the cell cycle (e.g., Smulders et al.
1994). Depending on the species, in the radicle only 2C
nuclei may be observed (Cichorium endivia, Capsicum
annuum), while in others (Castanea sativa, Hordeum
vulgare), besides the 2C nuclei, a small number of 4C DNA
nuclei have also been identified (Bino et al. 1993; Gendreau
et al. 2008). For example, in the H. vulgare embryo, 82% of
the nuclei of the radicle tip cells have a 2C content and only
18% have a 4C DNA content indicating that the majority of
cells have been arrested in the G1 phase of the cell cycle.
Interestingly, the percentages of 4C nuclei were different in
the plumule (22%) suggesting that the cell cycle activity in
the developing embryo has stopped at different stages
depending on the organ (Gendreau et al. 2008). The occur-
rence of endopolyploidy (8C DNA nuclei) in the embryo has
also been reported in some species including Phaseolus
vulgaris, Spinacia oleracea, and Chenopodium quinoa.
This indicated that endopolyploidisation can take place in
the early stages of tissue differentiation during embryo devel-
opment. Most often the endopolyploid cells have been
observed in the radicles, which first appear during seed ger-
mination (Bino et al. 1993; Kolano et al. 2009).
7.4.4.2 Post-embryogenic Development
Systemic control of endopolyploidy has been shown in plant
species such as Arabidopsis thaliana, Cucumis sativus, Sola-
num lycopericum, and Mesembryanthemum crystallinum
(De Rocher et al. 1990; Galbraith et al. 1991; Gilissen
et al. 1993; Smulders et al. 1994). Polysomaty in these
J. Maluszynska et al.
species may be expressed in roots, leaves, petioles, stem
internodes and flowers. Increasing evidence has revealed
that a pattern of endopolyploidy is characteristic for tissue
type and developmental stage indicating that polysomaty
is spatially and temporally regulated. The patterns of
endopolyploidisation within a plant of one species are usu-
ally different in various organs and correlate with the devel-
opmental stage (Galbraith et al. 1991; Gilissen et al. 1993;
Barow 2006).
Seedling Development
During seed germination and subsequent seedling develop-
ment an increasing proportion of endopolyploid nuclei
appear. The highest level of endopolyploidy is usually
observed in the hypocotyls and primary roots (Fig. 7.5).
The extent of endopolyploidisation differs significantly
between species, for example hypocotyls of Solanum
lycopersicum, Brassica oleracea and Chenopodium quinoa
(quinoa) undergo two endocycles (up to 16C DNA) whereas
hypocotyls of Beta vulgaris (sugar-beet) undergo three
endocycles (up to 32C DNA) (Smulders et al. 1994; Kudo
and Kimura 2001a; Sliwinska and Lukaszewska 2005;
Kolano et al. 2009).
There are also species-specific endopolyploidy patterns in
primary roots. The number of endocycles can be as low as
one (e.g., in tomato roots) or more numerous as in sugar-beet
roots (Smulders et al. 1994; Sliwinska and Lukaszewska
2005). Older organs usually exhibit higher levels of endo-
polyploidy than younger ones. An increase of endopoly-
ploidy to 32C and 16C DNA has been observed in the
hypocotyls and primary roots of cucumber and cabbage
seedlings, respectively (Gilissen et al. 1993; Kudo and
Kimura 2001a). Another pattern of endoreduplication in
seedling development has been observed in quinoa and
sugar-beet. Here the endopolyploidy level initially increases
and then gradually decreases (Sliwinska and Lukaszewska
2005; Kolano et al. 2009).
The high level of endopolyploidy observed in hypocotyls
and primary roots can be explained by the considerable
proportion of vascular tissue present in these organs. The
increase of endopolyploidy seems to be correlated with their
rapid elongation during germination, and this may be
connected with vascular tissue development. Endopolyploid
cells appear slightly later in the cotyledons during germina-
tion and seedling development (e.g., cucumber, tomato,
quinoa, sugar-beet). The level of endopolyploidy depends
on the species and can reach 4C in C. quinoa (Fig. 7.5), 32C
in A. thaliana or as much as 64C DNA in Lupinus cosentinii
(Galbraith et al. 1991; Hajdera et al. 2003; Kolano et al.
2009). A very high level of endopolyploidisation in
L. cosentinii cotyledons seems to be typical for seed storage
organs, and could be connected with the high metabolic
activity of the tissue (Larkins et al. 2001).
7 Endopolyploidy in Plants 111

960 a 2C b 2C
640

640 480

320
320
160 4C
4C
0 0
1 10 1 10
480
c 2C 4C
d 2C

320 480 4C

320
160
8C
160
0
1 10
0
4C 1 10
e 2C
240

160

80 8C
16C
0
1 10

Fig. 7.5 Evaluation of endopolyploidy in seedling of Chenopodium quinoa (quinoa) determined by flow cytometry. (a) Leaf; (b) shoot apex;
(c) hypocotyl; (d) cotyledon; (e) root. x-axis nuclear log DAPI fluorescence intensity, y-axis number of nuclei

Species- and Organ-Specific Endopolyploidisation
Almost all plant organs can become polysomatic during
vegetative and reproductive development. However, an
absence of endopolyploidy in the shoot and root apical
meristems is generally observed (Kudo and Kimura 2001a;
Lim and Loh 2003). It has been suggested that repression of
endopolyploidisation at the shoot apex might be one of the
mechanisms to ensure genetic stability of the germ line
(Kudo and Kimura 2001a). Usually the frequency of endo-
polyploid nuclei is highest in organs rich in vascular tissue
(e.g., petioles, leaf main midribs, stem and root) as well as in
storage tissue (cotyledons, endosperm) (Barow and Meister
2003; Barow 2006).
The level of endopolyploidy is generally species- and
organ-specific but usually older organs exhibit a higher
level of endopolyploidy than younger ones. An exception
to this may be the leaves of Cucumis sativus where the
pattern of polysomaty remains unchanged in young and
mature leaves (Gilissen et al. 1993). In the same organ, a
gradient of endopolyploidy can also be observed. For
example, in Amaranthus caudatus the number of cells with
2C DNA decreased and the number of cells with a DNA
content higher than 4C increased in the basal part of the
shoot compared with the youngest most distal part (Fig. 7.6)
(Kolano et al. unpublished). In the leaves of the orchid
Vanda ‘Miss Joaquim’ the level of endopolyploidy increased
from base to tip (acropetally), while in cotyledons of
C. sativus and leaves of Spathoglottis plicata, it increased
from tip to base (basipetally) (Gilissen et al. 1993; Yang and
Loh 2004).
The level of endopolyploidy in leaf cells differs consider-
ably among species. For example, cells with a DNA content
higher than 4C were not observed in the young fully extended
lamina of Chenopodium quinoa (Kolano et al. 2009) whereas
in leaves of Arabidopsis thaliana or Solanum lycopersicum
cells were present that had undergone one or two endocycles.
Interestingly, the leaves of succulents such as Portulaca
grandiflora or Mesembryanthemum crystallinum can exhibit
high levels of endoreduplication up to 64C. It has been
suggested that the high nuclear DNA content in succulent
112
Fig. 7.6 Comparison of endopolyploidy patterns in young and old
organs of Amaranthus caudatus. (a) Young leaf lamina; (b) young
leaf petiole; (c) young internode of the shoot; (d) old leaf lamina,
(e) old internode of the shoot; (f) old leaf petiole. x-axis nuclear log
DAPI fluorescence intensity, y-axis number of nuclei
leaves occurs in the water storing mesophyll cells (De
Rocher et al. 1990; Mishiba and Mii 2000).
Tissue-Specific Endopolyploidisation
Differences in the pattern of endopolyploidy can occur
within an organ. For example, in sugar-beet leaves there
was no endopolyploidisation in any part of the leaf lamina,
regardless of leaf age. However, endopolyploid nuclei
occurred in the main midrib (Lukaszewska and Sliwinska
2007). Even within a single tissue, the cells can differ in
endopolyploidy level. An analysis of Brassica species
showed that in the distal part of a petal nuclei were small
and numerous with DNA contents of 2C and 4C, whereas in
the proximal part nearly 40% of the epidermal cells were
large and extremely elongated, with endopolyploidy up to
32C (Kudo and Kimura 2002). An additional example is
found in the epidermis of Arabidopsis. Here a regular pattern
of endopolyploidy is evident in the epidermal cells, with the
nuclei of epidermal pavement cells exhibiting 2C, 4C, and
8C DNA amounts in the stem epidermis and 2C–16C in the
leaf epidermis. Leaf trichome nuclei also have elevated
ploidy levels of 4C, 8C, 16C, 32C and 64C. In contrast, the
guard cell nuclei have a 2C DNA content i.e., typical of cells
in the G1 phase of the cell cycle. Interestingly, among
epidermal pavement cells a gradual decrease in endopoly-
ploidy was found towards the guard cells (Melaragno
et al.1993).
Endopolyploidy in Flowers
Endopolyploidy occurs in most flower organs such as the
petals, sepals, carpels and stamens of many species (Mishiba
J. Maluszynska et al.
and Mii 2000; Kudo and Kimura 2001b; Barow and Meister
2003; Lukaszewska and Sliwinska 2007). The presence of
endopolyploid cells has been shown in different organs of
the cabbage flower. Filament tissue contained cells with six
ploidy levels (2C–64C) whereas petals had cells with five
ploidy levels (2C–32C). A 2C–8C range of ploidy levels in
the carpel tissue has been described while a high level of
endopolyploidy has been reported for stamens (Kudo and
Kimura 2001b).
For many species the highest level of endopolyploidy has
been shown to occur in the flower organs (Barow and Meis-
ter 2003). For instance, petals of the orchids Oncidium
varicosum and Phalaenopsis spp. and cabbage (Brassica
oleracea) have been shown to undergo extensive endopoly-
ploidisation with some cells reaching 64C (Kudo and
Kimura 2001b; Lee et al. 2004). However, this is not always
the case as in some species such as the generally polysomatic
plant A. thaliana, petal cells remain diploid (Galbraith
et al. 1991).
Endopolyploidy in Endosperm
The endosperm is the main source of nutrition for
germinating seeds. Endosperm formation starts soon after
fertilisation and it is often associated with the switch from a
mitotic cell cycle to an endocycle. In maize, up to 90% of the
nuclei can undergo endoreduplication but the number of
endocycles varies among endosperm cells. The cells in the
center of the endosperm enter endoreduplication first and are
followed by the most adjacent cells and so forth. The spatial/
temporal pattern of the cell cycle/endocycle switch creates a
gradient in the DNA content and cell size with the smallest
nuclei (3C and 6C) located at the periphery of the endosperm
while increasingly larger endopolyploid nuclei are found
towards the central region. Most commonly, four to five
endocycles occur although individual nuclei with a DNA
content of up to 690C have been observed. This endopoly-
ploidisation is correlated with an increase in endosperm cell
size and the rapid synthesis of starch, suggesting that endo-
polyploidy can increase metabolic activity, rRNA synthesis
and transcriptional activity. However, a 50% reduction in the
mean DNA content of mutant maize endosperm has very
little affect on the accumulation of starch and storage
proteins. Therefore, it is possible that endopolyploid endo-
sperm may simply provide a mechanism to store nucleotides
during the development and germination of the embryo
(Larkins et al. 2001; Leiva-Neto et al. 2004; Lee et al. 2009).
Endopolyploidy in Fruit
The input of endopolyploidisation in the development of
fruit has been extensively studied in the tomato (Solanum
lycopersicum—see review by Chevalier et al. 2011). Growth
of the tomato fruit starts after the bloom with intensive cell
divisions. As development proceeds, the proliferative
7 Endopolyploidy in Plants
activity of the cells slows down until it ceases and the
population progressively enters the stage of cell enlargement
(Bertin et al. 2003). Cessation of cell divisions and an
increase in cell size are closely linked to endopolyploi-
disation (Melaragno et al. 1993; Traas et al. 1998; Joubes
and Chevalier 2000). Endopolyploid cells are present in the
pericarp, locular gel and central columella at the breaker
stage. The three tissues display different ploidy profiles,
with the highest C-values (512C) in the pericarp. Sepals
also become endopolyploid during fruit growth, but to a
lower extent, with C-values up to 32C. Cheniclet et al.
(2005) reported a positive correlation between endoredu-
plication and cell size in the pericarp tissues of the tomato.
7.4.4.3 Endopolyploidy in the Nodulation Process
Endopolyploidisation is involved in the nodulation process
in legumes. These species establish a nitrogen-fixing symbi-
osis with rhizobia bacteria. During this process the root
nodule is formed to supply the host plant with nitrogen.
(e.g., Medicago truncatula–Sinorhizobium meliloti interac-
tion). A longitudinal section of a M. truncatula nodule
reveals cells at different stages of development, and exhi-
biting a differentiation gradient from the apical part to the
basal tissues attached to the root (Newcomb et al. 1979). The
apical meristem (Zone I) is composed of small proliferating,
uninfected cells. Endoreduplication is primarily associated
with the infection zone (Zone II). Indeed, the symbiotic
differentiation program involves the arrest of cell division,
followed by several rounds of endoreduplication. Enlarged
endopolyploid cells are invaded and host the bacteroids,
whereas the small diploid cells remain uninfected. Endo-
reduplication continues into Zone III where fully diff-
erentiated bacteroids develop the capacity to fix nitrogen.
The DNA content increases from 2C and 4C levels up to
64C and is accompanied by nuclear and cell enlargement
(Kondorosi and Kondorosi 2004; Wildermuth 2010).
Endoreduplication and cell enlargement seem to play a cen-
tral role in nodulation as studies of Medicago plants, whose
nodules had reduced endopolyploidy levels, exhibited inef-
ficient nitrogen fixation. The repeated endoreduplication
cycles during symbiotic cell development might have dual
roles, (1) they enable the extreme enlargement of cells to
host the bacteroids and (2) they provide an energy and
nutrient supply for the bacteroids through the increased
transcriptional and metabolic activities in the host cell
(Kondorosi and Kondorosi 2004).
7.4.4.4 Relationships Between DNA Content,
Nuclear Volume and Cell Size
The contribution and role of endopolyploidy to plant growth
and development is not yet fully understood. One problem
that needs explaining is the correlation between the nuclear
DNA content and cell size (Fig. 7.1). Plant development and
cell differentiation are usually accompanied by an increase
113
in cell size and since a correlation between nuclear DNA
content and cell size has been reported in A. thaliana and
other plant species (Melaragno et al. 1993; Bertin 2005;
Jovtchev et al. 2006) this has led to the hypothesis of a
‘nuclear–cytoplasmic ratio’. This hypothesis states that
some control mechanism ensures that the amount of cyto-
plasm in a cell is proportional to the amount of DNA in its
nucleus. Indeed, a simple comparison of genome size and
nuclear and cell volume among species supports this theory.
Species with larger genomes generally have larger nuclear
and cellular volumes and it is well known that polyploid
cells are bigger than diploid cells (Cavalier-Smith 2005;
Jovtchev et al. 2006; Webster et al. 2009).
The hypothesis that endoreduplication does indeed deter-
mine the rate of cell growth has been proposed based on
numerous observations. For example, cells that have
undergone endopolyploidisation are larger than comparable
diploid cells as has been shown in the epidermis of
A. thaliana and Brassica species (Melaragno et al. 1993;
Kudo and Kimura 2002; Jovtchev et al. 2006). In addition,
a link between cell size and the average C-value has been
reported in seeds (Lemontey et al. 2000) and fruits (Bertin
2005). The hypothesis is further supported by the fact that
endoreduplication usually precedes cell expansion (Traas
et al. 1998). For example, endoreduplication seems to be a
prerequisite for cell expansion during the growth of tomato
fruit (Bertin 2005). Endoreduplication is also associated
with the increase in cell size as observed in A. thaliana leaf
epidermal cells, etiolated hypocotyls and various mutants
that exhibit a truncated size and/or morphology (Melaragno
et al. 1993; Gendreau et al. 1997; Sugimoto-Shirasu and
Roberts 2003). However, the ‘nuclear–cytoplasmic ratio’
theory does not explain why cells from different tissues in
a given organism with the same amount of DNA differ in
nucleus and cell size. For example, different relationships
between the endopolyploidy level and cell volume were
found in the root cortex of 18 accessions of A. thaliana
(Beemster et al. 2002). In addition, although endopolyploi-
disation clearly contributes to increasing cell size, a key
feature of this phenomenon seems to be a boost in the
cell’s capacity for future growth as the determination of
the final cell size also depends on the development and
function of the cell. The current data therefore actually
argue for a physiological role of endopolyploidy as a facili-
tator of cell growth and as an accelerator for organ growth
(Kondorosi et al. 2000; Chevalier et al. 2011).
7.5 Polysomatic and Non-polysomatic
Plants in Culture In Vitro
Plant organs, or their fragments, are frequently used as a
source of explants for tissue and cell culture in vitro and
for genetic transformation experiments. Explants from
114 J. Maluszynska et al.

polysomatic plants are composed of a heterogeneous popu- cultures derived from polysomatic plants. He also noted
lation of endopolyploid cells (mixoploid). Such non- that such plants were cytologically unstable and all contained
dividing, differentiated cells can be induced into mitosis by aneuploid cells and cells with a reduced number of
wounding or phytohormone treatment. In the conditions of chromosomes compared with the donor plants. In contrast,
an in vitro culture endopolyploid cells undergo dedifferenti- callus derived from non-polysomatic plant explants were
ation and start mitosis thus displaying polyploid chromo- observed to be chromosomally stable over long periods of
some numbers. These polyploid cells are one of the major time in in vitro culture, remaining diploid and never
sources of chromosomal instability in a tissue culture and undergoing endoreduplication. He thus considered that it
somaclonal variation in regenerated plants. In contrast, was the presence of preexisting endopolyploidy that seemed
explants of non-polysomatic plants contain only diploid to be crucial for a lack of chromosome stability in a cell or
cells and are thus chromosomally stable during in vitro tissue culture and that this may also play a role in contributing
culture. to the undesirable somaclonal variation in regenerated plants.
The relationship between the type of primary explant and
the occurrence of chromosomal variation during callo-
genesis, long-term callus culture and capability to regenerate
7.6 Methods to Analyse Endopolyploidy
has been investigated in two model plants (1) the non-
polysomatic species Crepis capillaris (2n ¼ 2x ¼ 6 þ Bs)
Endopolyploidy can be studied both quantitatively and qual-
and (2) the polysomatic Arabidopsis thaliana (2n ¼ 2x
itatively. Quantitative methods allow the ploidy pattern of a
¼ 10 and 2n ¼ 4x ¼ 20). Stability at the diploid level
tissue, organ or entire organism to be established. For this
during 1 year of C. capillaris callus culture has been
purpose flow cytometry is the most widely applied technique
reported by several authors, although structural chromo-
although image cytometry (ICM) is also generating
somal aberrations did occur at the diploid level. Polyploi-
increased interest (Vilhar et al. 2001). Both techniques
disation occurred in the older callus culture but regenerated
enable the fast, high-throughput analysis of nuclear DNA
plants were only diploid (Maluszynska 1990). Stability at the
content. Sample preparation for both techniques requires the
diploid level has also been reported for callus or cell suspen-
isolation of a nuclear suspension and this is usually achieved
sion cultures for other non-polysomatic species such as
by the mechanical homogenisation of a tissue in an appro-
Helianthus annuus (Butcher et al. 1975), Lilium longiflorum
priate isolation buffer. Afterwards, the nuclei are stained
(Sheridan 1975) and the gymnosperm Pinus nigra (Papes
with a fluorochrome, most often DAPI or propidium iodide,
et al. 1983).
and their DNA content is evaluated on the basis of fluores-
A cytogenetic analysis of A. thaliana leaf explants during
cence intensity (Doležel and Bartos 2005; Doležel et al.
callogenesis showed that the first mitotic polyploid cells
2007). Data are visualized in the form of a histogram in
occurred just after the third day of culture. Flow cytometric
which the consecutive peaks represent rounds of endoredu-
analysis revealed additional endoreduplication cycles up to
plication (e.g., Figs. 7.5 and 7.6). The mean value of
64C (Fras et al. 2007). In primary callus cultures derived
endopolyploidisation can be calculated based on the histo-
from different explants, callus lines of meristematic origin
gram, which enables a comparative analysis of endopoly-
(i.e., root tips) were characterized by a narrower range of
ploidy between different samples.
ploidy levels (2x–10x, i.e., 10–50 chromosomes) than callus
There are two parameters that determine the endopoly-
lines of non-meristematic origin (leaves, cotyledons,
ploidy mean value: (1) the mean C-level and (2) the cycle
hypocotyls) which had ploidy levels ranging from 2x to
value (Barow and Meister 2003; Barow and Jovtchev 2007).
15x (10–75 chromosomes) although cells with 2x and
The mean C-level indicates the mean DNA content per
8x were the most common). The frequency of polyploid
nucleus and is calculated from the number of nuclei at
cells increased with age of culture and was correlated with
each ploidy level multiplied by the corresponding ploidy
the type of explants (Fras and Maluszynska 2004). Among
level. The sum is divided by the total number of nuclei
A. thaliana regenerants a high frequency of polyploids,
being investigated up to the formula:
mostly tri- and tetraploids, have been reported (Sangwan
et al. 1992; Altman et al. 1994; Mittelsten Scheid et al.
1996). Correlations between the level of endopolyploidy of Mean C level ¼ ð2  n2C þ 4  n4C þ 8  n8C
the plant organ tissue donor for in vitro culture and the þ :::Þ=ðn2C þ n4C þ n8C þ :::Þ
frequency of polyploid regenerants were described for Sola-
num lycopersicum (Van den Bulk et al. 1990). The cycle value represents the mean number of
D’Amato (1986) described several processes like endocycles per nucleus and is calculated from the number
endoreduplication, endomitosis and fusion of nuclei as the of nuclei at each ploidy level multiplied by the number of
mechanisms responsible for polyploidisation in tissue endoreduplication cycles necessary to reach the
7 Endopolyploidy in Plants
corresponding ploidy level. The sum is then divided by the
total number of nuclei according to the formula:
Cycle value ¼ ð0  n2C þ 1  n4C þ 2  n8C þ 3
 n16C . . .Þ=ðn2C þ n4C þ n8C þ n16C . . .Þ
A cycle value below 0.1 indicates a non-polysomatic type
of plant.
Although flow cytometry enables the ploidy levels in
large numbers of cells to be measured rapidly, it does not
provide any tissue-specific information. The advantage of
ICM over flow cytometry is that it combines cytometric data
with fluorescent microscopy (Vilhar et al. 2001). Nuclei are
investigated on microscopic slides and after (or simulta-
neously with) the determination of the DNA content, they
can be used for studies with other techniques such as fluo-
rescence in situ hybridization (FISH) or immunodetection
(Fig. 7.4c).
Insights into the organization of chromatin in endo-
replicated nuclei can be obtained by FISH which is useful
not only to determine the ploidy level in a single nucleus of
the tissue being analysed but can also provide information
about the mechanisms by which endoreduplication is driven
(Weiss and Maluszynska 2001). Coupling FISH with
cytometric data allows information about the organization
and structure of endoreplicated chromosomes at various
ploidy levels to be obtained (Schubert et al. 2006, 2008).
In vivo methods using transgenes with green fluorescent
protein (GFP) linked to cell-type specific promoters are
very promising for endopolyploidy studies as they enable
the C-value status of specific cell types to be analysed
(Zhang et al. 2005; Galbraith 2007). Such approaches have
the potential to probe the onset, progression and regulation
of endopolyploidy at a much higher resolution than has
hitherto been possible. However, so far this approach has
only been used for studies of A. thaliana (Mathieu et al.
2003; Fang and Spector 2005; Matzke et al. 2005). Never-
theless, its application to other plants which can be
transformed seems likely to increase as such techniques
become more widely applicable to other systems.
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 Meiosis: Recombination and the Control
of Cell Division 8
Eric Jenczewski, Raphael Mercier, Nicolas Macaisne, and Christine Mézard

Contents 8.1 Introduction

8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Meiosis is a key step in the reproduction of many species,
8.2 Meiotic Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
8.2.1 Initiation of Meiotic Recombination . . . . . . . . . . . . . . . . . . . . . . 123 halving ploidy levels to produce haploid gametes, which are
8.2.2 Meiotic DNA Double-Strand Break Repair . . . . . . . . . . . . . . . 124 then restored by fertilization. The reduction in ploidy in
8.2.3 Crossover Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 meiosis results from one cycle of replication followed by
8.2.4 Crossover Formation and Interference . . . . . . . . . . . . . . . . . . . . 127 two cell divisions (Fig. 8.1). During the first division, which
8.3 Dynamics of Chromosomes, Chromatin Structure is very specific to meiosis, the homologous chromosomes
and Meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 segregate to opposite poles. The second division during
8.4 Meiotic COs in Polyploid Plants . . . . . . . . . . . . . . . . . . . . . . . . 129 which the sister chromatids separate, is a modified version
8.4.1 Changes in CO Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 of mitosis. Thus a diploid cell produces four haploid spores.
8.4.2 Genetic Control of CO Formation . . . . . . . . . . . . . . . . . . . . . . . . . 130 However, depending on species-specific mechanisms
8.5 The Meiotic Cell Cycle in Plants . . . . . . . . . . . . . . . . . . . . . . . . 131 for sporocyte maturation, it is possible that not all spores
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 become gametes. For example, in Arabidopsis thaliana four
spores are obtained from female meiosis but only one
matures to produce an embryo sac containing an egg cell,
while in male meiosis, all four spores give rise to pollen
grains.
During the first meiotic division, homologous chromo-
somes recombine producing mosaic chromosomes that
are harmonious patchworks of the original genetic material
contained in the meiocytes. The underlying molecular
mechanisms for these recombination events are very well
conserved among species even if each species has shown
some specificity in regulation of the pathways or the molec-
ular actors involved. Recombination is initiated by the
formation of programmed DNA double strand breaks
(DSBs) (Fig. 8.2a). These breaks are repaired using mainly
the homologous chromosome as a template. The repair
process leads either to a crossover (CO, i.e., reciprocal
exchange of a large fragment of the chromosome) or a
gene conversion not associated with a CO (non-reciprocal
exchange of small DNA fragments, i.e., a non-crossover,
NCO)(see Fig. 8.2).
In this review we summarize and discuss recent findings
concerning the main steps of chromosome recombination
C. Mézard (*)
Institut Jean-Pierre Bourgin, Institut National de Recherche
and segregation during meiosis and the control of cell
Agronomique, route de Saint-Cyr, 78026 Versailles, cedex, France division in diploid and polyploid plants. Several excellent
e-mail: Christine.Mezard@versailles.inra.fr

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 121

DOI 10.1007/978-3-7091-1160-4_8, # Springer-Verlag Wien 2013
122 E. Jenczewski et al.

G2/Prophase
S phase

Metaphase

G1

Anaphase

G1
Telophase
b

G1 S phase G2/Prophase I
Metaphase I

Anaphase I

Telophase I

Prophase II

Metaphase II

Anaphase II

Telophase II

Fig. 8.1 Schematic representation of (a) mitosis and (b) meiosis. In replication followed by two rounds of chromosome segregation. The
the mitotic cell cycle: a “mother” cell (2n) produces two daughter cells first division is very specific to meiosis and it segregates homologous
(2n) that can re-enter the mitotic cycle. In contrast, in meiosis the chromosomes; the second round is a modified version of the mitotic
“mother” cell (2n) produces four haploid (n) spores after one cycle of division, sister chromatids are sent to opposite poles

reviews have been published recently (Hamant et al. 2006; we focus only on new aspects from recent results obtained in
Mercier and Grelon 2008; De Muyt et al. 2009a; Martinez- plants but also in other kingdoms to better understand the
Perez 2009; Ronceret et al. 2009; Harrison et al. 2010), thus meiotic process as a whole.
8 Meiosis: Recombination and the Control of Cell Division 123

AtSPO11-1; AtSPO11-2;
a AtPRD1; AtPRD2; AtPRD3;
AtPRD4; AtPHS1

AtDMC1; AtRAD51; ASY1;

c AHP2; AtMND1; SDS;
AtRPA1a

AtMUS81
AtEME1
d k n
AtMER3
AtZYP1
AtZIP4
AtRPA1a e o
AtMSH4
AtMSH5
SHOC1
PTD f p
AtMLH1
AtMLH3 l
BLM
i TOP3α q
BLAP75

g h j m r

Class I Class II
NCO NCO NCO
CO CO

Fig. 8.2 A simplified view of the meiotic recombination mechanisms.
(a) Recombination is initiated by the programmed formation of DNA
double strand breaks (DSBs). (b) DSBs are then processed to produce
30 single stranded DNA ends. (c) Next, DNA ends invade allelic sequences
on the homologous chromosomes. (d–f) A subset of these recombina-
tion intermediates form double Holliday junctions (dHJs). Resolution
of dHJs leads either to class I crossovers (CO) (g) or to non-crossovers
(NCOs) (h). (i, j) On the other hand, decatenation of dHJs produces
NCOs. (k, l, m) Nascent DSB partner complexes shown in (c) may also
be processed by Single Strand Annealing (SSA). After DNA invasion
and replication, the extended end is displaced from the template (k) and
anneals with its partner (l) producing a NCO (m). (n–q) Class II COs
are produced via a single Holliday Junction. Arabidopsis proteins
known (black characters) or thought to be (grey characters) involved
are indicated at their presumed step of action

A. thaliana, whereas AtSPO11-3 is not (Stacey et al. 2006).

8.2 Meiotic Recombination
8.2.1 Initiation of Meiotic Recombination
In all species, the topoisomerase-like protein Spo11 generates
meiotic DSBs (Keeney 2007). Three Spo11 homologs are
present in Arabidopsis thaliana, Oryza sativa (rice) and
several other plant genomes (Malik et al. 2007). AtSPO11-1
and AtSPO11-2 are both necessary for DSB formation in
In rice, functional analysis of OsSPO11-1 demonstrated its
role in DSB formation (Yu et al. 2010). In Saccharomyces
cerevisiae (budding yeast), six other meiotic proteins
(Rec102, Rec104, Rec114, Ski8, Mer2 and Mei4) and three
proteins, also involved in DSB repair in vegetative cells
(Rad50, Mre11, Xrs2), play a role in meiotic DSBs formation.
In fission yeast Schizosaccharomyces pombe, in addition
to Spo11, six genes that have no obvious homologs in
S. cerevisiae participate in DSB formation (Mde2, Rec6,
124
Rec10, Rec15, Rec24) (Keeney 2007). In plants as in other
eukaryotes, several screens (forward or reverse, see Mercier
and Grelon 2008) were developed to identify genes that
control this DSB formation step. It appears that only a few
of these additional DSB-forming proteins are conserved in
other kingdoms and even when they show sequence
similarities their function in DSB formation may not be
entirely conserved. For example, in A. thaliana, the DSB
processing role of Rad50 and Mre11 is only conserved in
vegetative cells and not in meiotic DSB formation (Bleuyard
et al. 2004; Puizina et al. 2004). The Arabidopsis Ski8
ortholog does not appear to play a role in DSB formation
(Jolivet et al. 2006). Nevertheless, two plant DSB formation
genes were successfully identified in forward screens:
AtPRD1 in Arabidopsis and PAIR1 in rice (Nonomura et al.
2004; De Muyt et al. 2007).
To improve the identification of new genes involved in
DSB formation in A. thaliana, a very efficient high through-
put analysis forward screen was designed (De Muyt et al.
2009b). A collection of 55,000 mutant lines was succes-
sively screened for (1) a reduction in fertility (1,280 lines)
then (2) meiotic defects (80 lines) and finally (3) meiotic
DSB formation defect-like phenotypes (28 lines). Following
fine localization, out of the nine loci identified, three new
genes AtPRD2, AtPRD3 (ortholog of PAIR1 in rice) and
AtPRD4 (Mathilde Grelon, personal communication) were
obtained that are involved in DSB formation. Several mutant
alleles of each gene were identified, suggesting that screen
saturation has been reached and that all genes required for
DSB formation have been identified in Arabidopsis (unless
there is gene duplication, preventing its identification by
forward genetics). Although at first sight no obvious
orthologs for these proteins could be identified outside the
plant kingdom, improved bioinformatics analysis then
showed that AtPRD2 could be the ortholog of Mei4 in
mice and yeast (Kumar et al. 2010). For AtPRD3 and
AtPRD4 as yet no significant homology has been identified
outside the plant kingdom.
The PHS1 gene was identified in maize and appears to be
involved in DSB repair but not formation and to prevent
synapsis between non-homologous chromosomes (Pawlowski
et al. 2004). When the Arabidopsis PHS1 homolog is inacti-
vated, nuclear import of the DSB repair protein Rad50 is
dramatically decreased. However, surprisingly, chromosomal
fragmentation was not reported, which is in contrast to the
phenotype of the single rad50 mutant (Bleuyard et al. 2004;
Ronceret et al. 2009). A recent report in mice suggested that
PHS1 is an ortholog of the Rec114 protein (see above;
(Kumar et al. 2010)). In S. cerevisiae, Rec114 interacts with
Mei4 and both proteins are involved in DSB formation
(Keeney 2007). In mice, the role of Mei4 in DSB formation
as well as its interaction with Rec114 is conserved suggesting
that its function in DSB formation may also be conserved.
E. Jenczewski et al.
It would be interesting to reanalyse the phs1 mutants in maize
and Arabidopsis with regards to these new data obtained in
mice concerning Mei4 and Rec114.
8.2.2 Meiotic DNA Double-Strand Break Repair
DSBs are then processed to produce 30 single stranded DNA
ends (Fig. 8.2b). In yeast, this single stranded DNA
interacts with RPA, a heterotrimeric protein that helps
the recombinase activity of Rad51 (reviewed in Sung
et al. 2003). Arabidopsis thaliana has five and Oryza sativa
(rice) has three homologs of the large subunit RPA1.
Analyses of the meiotic proteome of Brassica oleracea,
a close relative of Arabidopsis, showed that AtRPA1a, one
member of the AtRPA1 family, is potentially a meiotically-
expressed gene (Sanchez-Moran et al. 2005). OsRPA1a has
the most amino acid sequence similarities with AtRPA1a
and belongs to the same phylogenetic clade (Chang et al.
2009). However, chromosomal defects in the corresponding
mutants are very different. A mutation in OsRPA1a leads to
severe fragmentation of meiotic chromosomes in male
meiocytes consistent with a similar role in rice and yeast.
In contrast, no fragmentation is observed in atrpa1 but
crossover (CO) rates are dramatically lower (see below)
(Osman et al. 2009) suggesting that this protein functions
later in the DNA repair process. Thus, different RPA
complexes in rice and Arabidopsis could perform the vari-
ous RPA tasks during meiotic recombination (Chang et al.
2009).
Next, DNA ends invade allelic sequences on the homo-
logous chromosomes and a subset of these recombination
intermediates forms double Holliday junctions (dHJs)
(Fig. 8.2c–f). In yeast, it is thought that the meiotic dHJs
are mainly resolved toward the formation of COs (Hunter
2007). However, in Arabidopsis, recent results suggest that
some of the meiotic dHJs could mature via a different
pathway (Chelysheva et al. 2008; Hartung et al. 2008).
A protein complex known as BTB in mammals and RTR
in yeast has anti-CO activity through the dissociation of dHJ
(or potentially aberrant dHJ) (Raynard et al. 2006). This
complex contains a RecQ-like helicase (BLM for Bloom in
Human, Sgs1 in yeast), a topoisomerase (Top3a) and a third
protein Blap75/Rmi1/Nce4, identified through its interac-
tion with either BLM or Sgs1. However, limited data are
available on the role of this complex in meiosis. In
Arabidopsis, a mutation in BLAP75 or a hypomorphic
mutation in Top3a results in extensive fragmentation of
meiotic chromosomes, which depends on meiotic DSB for-
mation, and an unusual arrest at the end of telophase I
(Chelysheva et al. 2008; Hartung et al. 2008). The activity
of BLM and RTR in vitro and in vegetative cells suggests
that at least in Arabidopsis, and potentially in all species,
8 Meiosis: Recombination and the Control of Cell Division
BLM/RTR could decatenate a subset of normal dHJs
or aberrant recombination intermediates containing dHJs
toward NCOs.
DNA DSBs are then repaired using either the homologous
chromosome or the sister chromatid as a template. In meiosis,
it is thought that there is a bias in the choice of template
and that repair is mainly directed toward the homolog. In
A. thaliana, AtDMC1 the meiotic recombinase, is one of the
major actors facilitating this bias (Couteau et al. 1999; De
Muyt et al. 2009b). Three other proteins also play a role in
this process (De Muyt et al. 2009b): ASY1, an axis associated
protein related to yeast Hop1 (Sanchez-Moran et al. 2007);
AtHP2, a homolog of yeast Hop2 which was suggested to
mediate the activity of the yeast Dmc1 protein in a complex
with AtMND1 (Chen et al. 2004; Vignard et al. 2007); and
SDS, an Arabidopsis cyclin that interacts with several Cyclin
Dependant Kinases (CDKs) (Azumi et al. 2002). The role of
PAIR2, the rice homolog of ASY1, in this process remains to
be elucidated (Nonomura et al. 2006).
The PAIR3 gene was also identified in rice in a large-
scale screen of a population of T-DNA insertional mutants
for partial or complete sterility (Yuan et al. 2009). The
protein has no similarity with any known proteins and no
known motifs except a coil-coiled domain. A preliminary
characterization of pair3 mutants found that synapsis
between homologous chromosomes at prophase I was
absent. Additional studies are needed to determine the role
of PAIR3 in meiosis.
The AtXRI1 gene is highly transcribed in response to
gamma rays (Dean et al. 2009). Detailed characterization
of atxri1 mutants showed that its chromosomes displayed
extensive fragmentation that did not depend on SPO11-
induced DSBs. Thus, AtXRI1 could play an important role
in the S-phase of meiosis together with two other proteins
which when depleted cause a similar phenotype, MEI1 and
CDC45 (Grelon et al. 2003; Stevens et al. 2004). AtXRI1
could also function in meiotic DSB repair by interacting
with MIP1, which in turn interacts with the AtMND1 protein
partner of AtHP2 (see above). Thus AtXRI1 could be
involved in various aspects of homologous replication:
repair of collapsed replication forks and post-replication
repair pathways. Finally, AtXRI1 is also involved in the
post-meiotic stages of pollen development.
8.2.3 Crossover Distribution
DSBs can be repaired either as COs or NCOs (Fig. 8.2). In
plants, as in many higher eukaryotes, it is thought that the
number of NCOs largely exceeds the number of COs. How-
ever only indirect evidence supports this supposition, no
direct test to measure NCO rates is presently available in
plants (reviewed in De Muyt et al. 2009a).
125
The number of COs per chromosome and per meiosis is
tightly controlled. In most species, there is a need for one
obligatory CO per pair of homologous chromosomes. More-
over, the distribution of COs along chromosomes is not
homogenous. Interference (see below) plays a role in both
controlling and constraining the final CO distribution but
additional factors are also important in localizing COs. In
all species with an available genetic map, the CO rate drops
in centromeric regions with estimated decreases between
five- to more than 200-fold depending on organisms (Talbert
and Henikoff 2010). Nevertheless, intriguing recent results
in maize suggest that centromeres are not inert for meiotic
recombination. In a mapping population, two gene conver-
sion events not associated with COs were mapped in
53 inbred lines giving a theoretical rate of 105 conversion
events per marker and per generation. This suggests that CO
formation in centromeric regions is not prohibited through
recombination abolition (e.g., prevention of DSB formation)
but by specific control of the DSB repair process. A precise
analysis of the frequency and distribution of NCOs is clearly
needed to better understand their rate of formation and
localization in plant genomes.
GC content is positively correlated with high CO rates in
many species such as rat, mice, human, zebra finch, honey-
bee, maize, even at a broad scale (Jensen-Seaman et al.
2004; Beye et al. 2006; Gore et al. 2009; Backstr€ om et al.
2010), although the underlying mechanisms responsible for
this correlation are still under discussion (see Duret and
Arndt 2008; Marsolier-Kergoat and Yeramian 2009). To
add a piece to the puzzle, in Arabidopsis, the variation in
CO rate of a male–female averaged recombination map was
negatively correlated to GC content (Drouaud et al. 2006).
Moreover, on the five Arabidopsis chromosomes, CO rates
negatively correlate with GC content in female meiosis but
not in male meiosis (Laurène Giraut, personal communica-
tion). Variation in CO rates also correlate with several other
genomic features such as transposable elements, the CpG
ratio, gene density, nucleotide polymorphisms or chromo-
some architecture properties like distance to telomeres or
centromeres (Petes 2001; Nachman 2002; Jensen-Seaman
et al. 2004; Myers et al. 2005; Saintenac et al. 2009). Never-
theless, none of these other characteristics are systematically
correlated with CO rate variation across every species. Thus
the various features that correlate with non-homogeneity in
CO rates may have causal relationships or may be inciden-
tally related.
CO rates and distribution can vary between male and
female meiosis in the same species. Haldane (1922) sugg-
ested that the heterogametic sex had a lower CO rate as a
consequence of selection against recombination between the
sex chromosomes. However, this hypothesis, referred to as
the Haldane and Huxley rule, has since been called into
question. Less recombination in the homogametic sex than
126
a f
b g
c h
d i
e j
Fig. 8.3 Prophase of the first meiotic division. Leptotene (a, f, k, p):
chromosomes begin to condense and the axial element (red line) along
chromosomes is formed (a, f); purple structures ¼ cohesin complexes;
red line: axial elements. (k) DAPI staining of an Arabidopsis meiocyte at
leptotene. (p) Immunolocalization of the ASY1 protein associated with
the axial element at leptotene. Zygotene (b, g, l, q): homologous
chromosomes synapse and the central element between the axial elements,
now called lateral elements, is formed to constitute the tripartite structure
called the synaptonemal complex (SC) shown in green in (b) and in
greater detail in (g). (l) DAPI staining of an Arabidopsis meiocyte at
the heterogametic one has been observed in some species
and heterochiasmy has been found without the presence of
sex chromosomes in plants such as Allium (Ved Brat 1966),
Brassica oleracea (Kearsey et al. 1995) and A. thaliana
(Armstrong and Jones 2001; Drouaud et al. 2007), and in
animals like the saltwater crocodile (Miles et al. 2009).
Other hypotheses have been proposed (reviewed in Hedrick
E. Jenczewski et al.
k p
l q
m r
n
o
zygotene. (q) Immunolocalization of the cohesin SCC3 protein at zygo-
tene. Pachytene (c, h, m, r): synapsis between homologous chromosomes
is complete (c, h). (m) DAPI staining of an Arabidopsis meiocyte at
pachytene. (r) Immunolocalization of the Zyp1 protein in the synap-
tonemal complex at pachytene. Diplotene (d, i, n): chromosomes
decondense and the SC is disassembled (d, i). (n) DAPI staining of an
Arabidopsis meiocyte at diplotene. Diakinesis (e, j, o): chromosomes
condense; bivalents individualize; homologous chromosomes remain
physically attached through chiasmata and sister chromatid cohesion
(e, j). (o) DAPI staining of an Arabidopsis meiocyte at diakinesis
2007) but none satisfactorily explain the variations in
heterochiasmy in all species.
Strikingly, a correlation was reported between CO num-
ber per chromosome and the total length of the synap-
tonemal complex (SC) (a proteinaceous structure that links
homologous chromosomes at the pachytene stage of meiosis I;
Fig. 8.3b, c). In A. thaliana male meiosis, there is a linear
8 Meiosis: Recombination and the Control of Cell Division
correlation between the mean CO number per chromosome
and the SC length (Laurène Giraut and Christine Mézard,
personal communication). Moreover, several studies have
shown that CO number and SC length vary coordinately,
even in situations where DNA length is constant (Kleckner
et al. 2003). For example, in human meiosis, males have
about half the CO number and total SC length compared
with females (Wallace and Hulten 1985). This correlation
was also reported in male and female meiocytes in the
flatworm Dendrocoelum lacteum (Croft and Jones 1989)
and zebrafish (Singer et al. 2002; Wallace and Wallace
2003) and in many other species with various individuals
of the same population (Fox 1973; Quevedo et al. 1997;
Tease and Hulten 2004). The reasons for this correlation
are still poorly understood.
8.2.4 Crossover Formation and Interference
In many species, at least two CO pathways coexist (Fig. 8.2).
Class I COs are sensitive to interference whereas Class II are
not. These two pathways also exist in plants (Copenhaver
et al. 2002). The phenomenon of interference was first
described at the beginning of the twentieth century as a
lower frequency of double COs in disjoint chromosomal
intervals than was expected if they occurred independently
(Sturtevant 1915). One of the consequences is that the physi-
cal distance between adjacent COs increases. When interfer-
ence is total as in Caenorhabditis elegans, only one CO
occurs per pair of homologous chromosomes. The mecha-
nism that mediates interference is still poorly understood.
Nevertheless, many molecular actors that are involved in
this Class I pathway have been identified. In the yeast
S. cerevisiae, interfering COs depend on the ZMM proteins
(Zip1, Zip2, Zip3, Zip4, Msh4, Msh5 and Mer3), Mlh1 and
Mlh3 whereas non-interfering COs need the Mus81-Mms4
complex (reviewed in Lynn et al. 2007). In A. thaliana,
homologs of many of these genes have been characterized:
AtMSH4, AtMSH5, AtMER3, two AtZYP1, AtZYP4, AtMLH3,
AtMLH1 and AtMUS81 (Fig. 8.2). Since a detailed descrip-
tion of these genes was recently reviewed elsewhere (Mercier
and Grelon 2008; De Muyt et al. 2009a), here we will only
discuss the main conclusions.
In rice, a mutant in the MER3 homolog displays a
similar phenotype to the Arabidopsis mer3 mutants
described (Wang et al. 2009). The SHOC1 gene is also a
likely functional homolog of Zip2 (Macaisne et al. 2008).
SHOC1 interacts with PTD, an Ercc1 homolog, suggesting
a role for a XPF-Ercc1 complex in Class I CO formation
(Nicolas Macaisne, personal communication). Two Mms4
homologs were identified in Arabidopsis and the corres-
ponding proteins interact with AtMus81 (Geuting et al.
2009). However, the meiotic phenotype of corresponding
127
mutants was not reported. As yet, no Zip3 homolog has been
identified. In zmm mutants, CO rates drop down to a mini-
mum of 15% of the wild type level and residual COs do not
display interference, suggesting the co-existence of two CO
pathways. Even in the absence of both the ZMM and
AtMUS81 proteins, and as observed in S. cerevisiae, some
COs (5–7%) are still detected suggesting either the exis-
tence of a third CO formation pathway or the activation of a
pathway only active when the two main ones are absent.
Unlike observations in yeast, zmm mutants in Arabidopsis
do not show defects in synapsis between homologous
chromosomes suggesting that synapsis depends on preco-
cious recombination intermediates rather than Class I COs.
The Arabidopsis genome contains two Zip1 orthologs,
AtZYP1a and AtZYP1b. Plants where both ZYP1 genes
have been downregulated using interfering RNAs undergo
synapsis between non-homologous chromosomes and form
aberrant multivalents whereas chiasmata labelled by the
MLH1 proteins are only slightly reduced (Higgins et al.
2005). The Zip1 homolog of rice, ZEP1 also shows some
specificity compared with other organisms (Wang et al.
2010a). In its absence, chromosomes align along their
entire length but the SC is not formed and the number
of COs may increase slightly suggesting a role later in
meiosis.
In maize, a statistical approach was developed to analyse
the position of late recombination nodules, which are elec-
tron dense structures thought to designate all Class I and
Class II COs (Falque et al. 2009). This analysis demonstrates
that (1) both Class I and Class II coexist in maize; (2) the
proportion of Class II COs varies from 6% to 23% (with an
average of 15%) depending on the chromosomes. A similar
proportion of non-interfering COs was identified in two
other plants: 30% in tomato (Lhuissier et al. 2007) and
28% in rice (Wang et al. 2009). These values, together
with the approximately 15% found in Arabidopsis, seem to
be in the same range as the values found in S. cerevisiae
(7–12%) and mice (5–11%) (Broman et al. 2002; Malkova
et al. 2004; Guillon et al. 2005).
8.3 Dynamics of Chromosomes, Chromatin
Structure and Meiosis
During meiosis, chromosomes undergo a series of dramatic
structural changes. They condense, exchange DNA
segments, pair, synapse, decondense and move in the
nucleus. Probably due to this complexity, meiosis is a rela-
tively long process compared to mitosis. It takes from two
(in Triticum aestivum) to nine times longer (in Trillium
erectum) in some species (Bennett 1971). In Arabidopsis,
the meiotic process is three times longer than the mitotic cell
cycle (8.5 h) (Armstrong et al. 2003). Prophase of the first
128 E. Jenczewski et al.

SMG7
M-CDK activity
TDM SMG7
TAM TAM
OSD1 OSD1 TDM

time
End of
e

II

II
as

se

se

se

se

se
S-phase
ph

ha

ha

ha

ha

ha
S-

op

ap

ap

ap

ap
1/

Pr

et

An

et

An
G

M
2/

M
G

Fig. 8.4 Model of the control of meiosis progression. The red line
represents a not yet identified cyclin-cyclin dependent kinase (CDK)
activity that controls meiosis progression. This activity must reach a
certain level to allow the G2/prophase to metaphase I transition and
reach a peak at metaphase I and metaphase II. The metaphase to anaphase
transitions are ensured by a rapid decrease in CDK activity. However, at
the end of anaphase I, a certain level of cyclin-CDK activity must be
maintained to allow entry into meiosis II and to avoid premature meiosis
exit. Arabidopsis genes involved in the control of meiosis progression are
indicated above the graph at their presumed time(s) of action

meiotic division (Figs. 8.3 and 8.4) is the longest stage
reflecting its complexity (21.3 h), the remaining stages
occur relatively quickly (2.7 h).
In leptotene (Fig. 8.3a, f, k, p) chromosomes begin to
condense and the axial element along the chromosomes
is installed. At zygotene (Fig. 8.3b, g, l, q), homologous
chromosomes synapse and the central element between the
axial elements, now called lateral elements, is formed to
constitute the tripartite structure called the SC (see above).
Synapsis is complete at pachytene (Fig. 8.3c, h, m, r). In
most species, telomeres cluster during early zygotene at a
single site on the nuclear envelope and this structure, called
the “bouquet” resumes at the beginning of pachytene.
Recombination is initiated by the formation of DSBs in
leptotene, thus before synapsis, and is completed during
pachytene. At diplotene (Fig. 8.3d, i, n), the SC disappears
but homologous chromosomes remain linked to each other
by COs and sister chromatid cohesion (a proteinaceous)
structure deposited on chromosomes after replication that
keeps sister chromatids linked to each other) until metaphase I.
At diakinesis (Fig. 8.3e, j, o), chromosomes condense further
and move to form the metaphase plate (reviewed in Hamant
et al. 2006).
Thus chromosomal movement across the nucleus is essential
for recombination, pairing and synapsis. Live observations
in S. pombe and S. cerevisiae have provided evidence of
unexpected vigorous chromosome motility during prophase I.
In S. pombe, before karyogamy, telomeres cluster tightly
beneath the spindle pole body (SPB) while centromeres
detach from the SPB. During karyogamy, the two telomere
clusters fuse and after karyogamy, the diploid nucleus
elongates about three times and initiates violent oscillations
from one end to the other forming the “horsetail” nucleus
that will remain throughout prophase I. This movement is
driven by microtubules coming from the SPB and telomeres
clustering beneath the SPB located at the leading edge of
the moving nucleus (reviewed in Ding and Hiraoka 2007).
In S. cerevisiae, chromosomal dynamics were visualized
using a SC protein tagged with green fluorescent protein
(GFP) (Scherthan et al. 2007; Koszul et al. 2008). SCs
exhibit dramatic and continuous movements, crossing rela-
tively large distances in the nucleus and changing shape.
This motion seems to be driven by actin cables of the
cytoskeleton.
Maize is the first higher eukaryote where live imaging
of meiosis prophase has been possible (Sheehan and
8 Meiosis: Recombination and the Control of Cell Division
Pawlowski 2009). Similar to the observations in yeasts,
maize chromosomes move extensively during zygotene and
pachytene with dynamic deformation of the nuclear enve-
lope. Chromosome motility is dependent upon the actin
and tubulin cytoskeletons. It has been proposed that these
chromosome movements either promote pairing of homolo-
gous chromosomes and/or eliminate unwanted connections
(Ding and Hiraoka 2007; Koszul et al. 2008; Sheehan and
Pawlowski 2009).
Chromatin structure has an important role to play in the
meiotic process. The most striking evidence has come
recently from mice and S. cerevisiae where a link between
trimethylation of the histone H3 at lysine 4 (H3K4) and the
formation of DSBs was demonstrated (Borde et al. 2009;
Baudat et al. 2010; Myers et al. 2010; Parvanov et al. 2010).
In both species, this trimethylation, which is associated with
an open chromatin structure, occurs before the formation of
DSBs (Borde et al. 2009; Buard et al. 2009). Set1 ensures it
in yeast. In mice, Prdm9 does the job: it is a meiosis-specific
protein containing a Set1-like domain and a series of
Zinc fingers that gives it sequence specificity for H3K4
trimethylation and thus DSB formation. No clear orthologs
of these proteins exist in plants and the similarity of
mechanisms remains to be demonstrated. However, recently,
in maize, based on the parallel between Mu transposon
insertion sites and regions with high recombination rates, it
was suggested that meiotic recombination occurs in regions
marked with open chromatin features in vegetative cells
such as a high level of trimethylated H3K4, trimethylated
H3K36, acetylated H3K9 and low level of trimethylated
H3K27 (Liu et al. 2009). Whether this correlation holds
true when DSB forming regions are looked at more precisely
remains to be confirmed and whether the mechanisms are
similar to those described in budding yeast and mice needs to
be elucidated.
In S. pombe (fission yeast), a mutation in the Set1 homolog
that severely reduces the level of trimethylated H3K4 does not
affect sporulation and thus probably not the global level of
DSBs (Noma and Grewal 2002). However in fission yeast,
histone acetylation plays a role in meiotic recombination
(Yamada et al. 2004). Histone acetylation is also correlated
with meiotic recombination in S. cerevisiae (budding yeast)
(Mieczkowski et al. 2007) even if it is now clear that it is not
the main player linked to DSB formation. Recent results
obtained in A. thaliana suggest that histone acetylation plays
a role in the meiotic process (Perella et al. 2010). In a mutant
line over-expressing the GCN5-related histone N-
acetyltransferase, the distribution of meiotic COs appeared
to be altered. Overall, the number of COs per meiotic cell
was similar to wild type but the number of COs per chromo-
some was altered with less COs on the longest chromosome 1
and more COs on the shortest chromosome 4. Sister chromatid
separation during the second meiotic division was also
129
disrupted suggesting that histone hyperacetylation may have
a profound effect on global chromatin structure affecting
different aspects of the meiotic process.
8.4 Meiotic COs in Polyploid Plants
It has now been clearly established that all flowering plants
have experienced at least one, and usually more, rounds of
whole genome duplication (i.e., polyploidy events) during
their evolution (Soltis et al. 2009; Van de Peer et al. 2009;
Fawcett et al. 2013, this volume). Nearly 30% of extant
flowering plants, including most of the world’s important
crops, are polyploids (Wood et al. 2009) while all other
angiosperms have been impacted by ancient polyploidy
events (they are palaeopolyploid species; Jiao et al. 2011).
One immediate consequence of polyploidy is an increase
in the number of chromosomes that can compete for pairing,
synapsis and recombination at meiosis. In newly-formed
synthetic or natural polyploids, the presence of more than
two partners usually leads to abnormal meiosis with multiple
or illegitimate chiasma associations that result in chro-
mosome missegregation, aneuploidy and partial fertility
(Ramsey and Schemske 2002). By contrast, extant wild
and cultivated polyploids display a clear tendency towards
a diploid-like meiotic behaviour. In autopolyploid species,
which are derived from within a single species (see Soltis
et al. 2010 for details), this results from an increased number
of bivalents forming at meiosis I, to the detriment of
multivalents, between any pair of homologs (resulting in
polysomic inheritance). In allopolyploid species, which usu-
ally arise via hybridization between different species
followed by chromosome doubling, regular meiosis requires
a non-random assortment of chromosomes into pairs with
COs being exclusively formed between homologous rather
than homoeologous chromosomes (i.e., inherited from dif-
ferent progenitors). Several reviews have examined
diploidization in polyploids from a cytological, genetic,
agronomic and evolutionary point of view (Jenczewski and
Alix 2004; Able and Langridge 2006; Hamant et al. 2006;
Able et al. 2009; Moore and Shaw 2009; Cifuentes et al.
2010a; Yousafzai et al. 2010a). We reiterate here that sup-
pression of COs between homoeologous chromosomes is
usually under polygenic control, with one locus having a
greater influence than the others and frequent gene-dosage
effects.
8.4.1 Changes in CO Distribution
An unexpected result that has emerged from comparisons of
CO formation in allopolyploid species and their diploid
progenitors is that COs sometimes get a boost in polyploids.
130
For example, Brubaker et al. (1999) compared the genetic
map of allotetraploid cotton with that of its diploid parents
and observed that allotetraploidy in Gossypium has been
accompanied by an increase in CO rates. However we do
not yet know if this observation holds true for polyploids in
general or if it only illustrates genotypic variation in the
propensity to form COs within the diploid species (i.e., it is
possible that the true parents of the allopolyploid cotton
formed more COs than the diploid plants used to build the
diploid genetic maps). Nevertheless, recent analyses in Bras-
sica which directly addressed this concern have confirmed
the trend observed in cotton. Leflon et al. (2010) analysed
the extent of CO variation in a set of related diploid AA,
allotriploid AAC and allotetraploid AACC Brassica hybrids
which were produced using exactly the same genotypes.
Combining immunolocalization of MLH1 and genetic
analyses, the authors observed that the number of COs in
the allotriploid AAC hybrid was higher than in the diploid
AA hybrid, with the allotetraploid AACC showing interme-
diate behaviour (Leflon et al. 2010). Although the precise
mechanisms involved are not presently known, these results
point towards an unexpected interplay between increased
ploidy and enhanced recombination. This in turn may tenta-
tively explain why increased and more variable recombina-
tion rates are observed in plants, which have been repeatedly
impacted by polyploidy, compared with animals (Gaut et al.
2007; Kejnovsky et al. 2009).
8.4.2 Genetic Control of CO Formation
8.4.2.1 Wheat
At present, most continuing efforts to identify genes
involved in the genetic control of recombination in
polyploids are focusing on wheat (Crismani et al. 2006;
Boden et al. 2007; de Bustos et al. 2007; Lloyd et al. 2007;
Khoo et al. 2008; Boden et al. 2009; Bovill et al. 2009; Perez
et al. 2010), and most notably on recombination between
homoeologous chromosomes (Griffiths et al. 2006; Al-Kaff
et al. 2008; Yousafzai et al. 2010b).
A number of these studies have aimed to identify wheat
orthologs of key recombination genes including Asy1 (Boden
et al. 2007), Msh7 (Lloyd et al. 2007), Mre11 (de Bustos et al.
2007) and Rad50 (Perez et al. 2010). The homoeologous
copies of Mre11 and Rad50 in wheat are highly conserved
but have slightly different expression levels (de Bustos et al.
2007; Perez et al. 2010); yeast two-hybrid assays revealed that
the products of the Rad50 and Mre11 genes show a great
affinity for one another that does not depend on their genome
of origin (Perez et al. 2010). This example demonstrates that
genetic redundancy in recent (<10,000 year-old) polyploids
provides a buffer effect for crucial functions.
Another approach has specifically aimed to identify novel
candidate meiotic genes. Crismani et al. (2006) produced and
analysed a large-scale transcriptomics dataset across a
E. Jenczewski et al.
meiotic time series in bread wheat and identified ~1,350
transcripts that were temporally-regulated during the early
stages of meiosis. Some of these candidates shared sequence
similarity with genes involved in chromatin condensation, SC
formation and recombination while most others showed no
sequence similarities with genes in the databases. Functional
characterization of these candidates has now to be pursued.
CO suppression between homoeologous chromosomes is
one expected additional constraint that an allopolyploid spe-
cies has to contend with during meiosis (compared to a
diploid species). In wheat, this control is ensured by a multi-
genic system (for review: Sears 1976; Jenczewski and Alix
2004), with the Ph1 locus (Wall et al. 1971) localized on the
long arm of chromosome 5B, having the most influence
(Riley and Chapman 1958; Sears and Okamoto 1958). This
system is efficient in both natural and synthetic wheats
(Mestiri et al. 2010) even when suppressors are present as
have been found in some genotypes of Aegilops speltoides
(Dvorak et al. 2006).
A 50-year quest was necessary to characterize the Ph1
locus at the molecular level (see Yousafzai et al. 2010b for a
review of the cloning efforts). Ph1 has been defined as a
region containing a cluster of seven genes related to cyclin-
dependant kinases (CDK) (see Sect. 8.5 for details about
these proteins) interrupted by a segment of heterochromatin
(Griffiths et al. 2006). This makes it distinct from its
homoeologs, which contain five (on chromosome 5A) and
two (on chromosome 5D) cdk-like genes, respectively
(Al-Kaff et al. 2008). Detailed bioinformatics and in silico
analyses has recently indicated that one of the Ph1 cdk-like
genes shares many functional attributes with human CDK2
(Yousafzai et al. 2010a). Human CDK2 promotes the timely
entry into premeiotic DNA replication and the first nuclear
division; it also induces transcription of a number of early
meiosis-specific genes including Hop1, which encodes a
component of the lateral element of the SC and plays an
important role in pairing of meiotic chromosomes
(Szwarcwort-Cohen et al. 2009). Expression profiling has
revealed that one 5A cdk-like and five of the Ph1 cdk-like
genes from the 5B locus are transcribed, including two
apparent pseudogenes (Al-Kaff et al. 2008). By contrast,
transcription of most functional cdk-like genes from 5A and
5D is detected only when the 5B copies are deleted. G. Moore
et al. have thus proposed that deletion of Ph1 could result in a
higher level of overall functional cdk-like proteins, which
may be responsible for the observed meiotic phenotypes of
ph1 mutants (Al-Kaff et al. 2008). If this hypothesis holds
true, then over-expression of cdk2-like target genes would be
expected in these mutants. Indeed, this is exactly what Boden
et al. (2009) observed; they showed that absence of Ph1
correlates with increased transcription of TaASY1, a gene
with significant sequence and functional similarity to Hop1.
Likewise, Knight et al. (2010) showed that okadaic acid, a
potent protein serine/threonine phosphatase inhibitor that
activates CDKs, promotes intergenomic recombination in
8 Meiosis: Recombination and the Control of Cell Division
wheat  rye interspecific hybrids, thereby phenocopying the
absence of Ph1. Even if direct functional evidence is still
required to demonstrate that wheat Ph1 is a functional ana-
logue of human CDK2, all current results indicate that it is
likely to be so (Yousafzai et al. 2010b). A major challenge of
the future will be to reconcile this function with all the
meiotic defects that have been observed in ph1 mutants:
non-resolution of incorrect and multiple early associations
between chromosomes (Holm and Wang 1988), CO forma-
tion between non-homologous chromosome segments (Luo
et al. 1996), defects in chromosome condensation and/or
scaffold organization (Mikhailova et al. 1998), and asynchro-
nous chromatin remodelling between homologs (Prieto et al.
2004; Colas et al. 2008).
8.4.2.2 Brassica napus
Over the last decade, Brassica napus has become a second
model for deciphering how COs between homoeologous
chromosomes are genetically suppressed. In this species, a
major locus, named PrBn for Pairing regulator in B. napus,
was shown to regulate CO formation between non-
homologous chromosomes in allohaploid plants produced
from different varieties (Jenczewski et al. 2003). Compara-
tive analyses between the meiotic phenotype of 363
allohaploids produced from 29 accessions and the maternal
origins of the same 29 accessions recently indicated that
these different levels of CO suppression between
homoeologs in B. napus allohaploids were the result of
repeated polyploidy in this species (Cifuentes et al. 2010b).
Actually PrBn shows incomplete penetrance (Cifuentes
et al. 2010b) and four to six additive and epistatic QTLs
have been detected, which contribute to the meiotic
behaviour of B. napus haploids (Liu et al. 2006). PrBn has
also been shown to determine the frequency, but not the
distribution of COs between homoeologous chromosomes
during meiosis of B. napus haploids and also to affect
homologous recombination in allotriploid AAC hybrids
(Nicolas et al. 2009). By contrast, genotypes with differing
PrBn activities at the haploid level do not change the rate of
CO formation between homologous chromosomes in allote-
traploid AACC hybrids (Nicolas et al. 2009) suggesting that
the effect of PrBn on recombination is dosage-dependent.
Experiments are ongoing to characterize the PrBn locus at
the molecular and phenotypic levels.
8.4.2.3 Cotton
Although it is clear that natural selection has certainly favored
the evolution of mechanisms that promote exclusive bivalent
formation in allopolyploids, it remains to be understood if the
CO suppression between homoeologous chromosomes is
paralleled by a similar effect on the other product of meiotic
recombination, i.e., NCO or gene conversion events. Recent
investigations in allotetraploid cotton have provided very
131
interesting information on this pending question (Salmon
et al. 2010). Tetraploid cotton is almost a strictly bivalent-
forming species (Kimber 1961) and displays disomic inheri-
tance (Reinisch et al. 1994; Brubaker et al. 1999) indicating
that COs are almost exclusively formed between homologous
chromosomes. Irrespective of this control, Salmon et al.
(2010) used a vast EST dataset to demonstrate that non-
reciprocal recombination events have occurred between
homoeologous sequences throughout polyploid divergence
and speciation in cotton. The authors first identified ESTs in
the allopolyploids derived from one parental EST but that
displayed at least one homoeoSNP from the other parent.
They subsequently sequenced de novo genomic DNA
corresponding to 20 putative recombination events and
validated 14 of these chimeric sequences. Six were sequences
from the D genome that have experienced conversion by the
A homoeolog, whereas four others exhibited the reciprocal
situation. Cloning and sequencing homoeologous copies from
a total of five allopolyploid cotton species demonstrated that
not a single recombination event was shared among species,
which indicated that these events occurred sporadically after
allopolyploid speciation.
8.5 The Meiotic Cell Cycle in Plants
Modification of the somatic cell cycle is one of the key
features of meiosis (N.B. meiosis is not a cycle, the products
of meiosis cannot undergo another round of meiosis, but by
analogy with mitosis we use the term cell cycle to describe
the control of progression through meiosis) (Figs. 8.1 and
8.4). The most prominent difference between meiosis and
the canonical mitotic cell cycle is that two divisions follow a
single replication at meiosis whereas replication and divi-
sion strictly alternate during mitotic proliferation. The mei-
otic cell cycle follows the same phases as mitosis: G1, which
precedes replication; S, the replication phase; G2, between
replication and nuclear membrane break-down; and a
mitotic phase during which the chromosomes contract and
align (prophase to metaphase) and separate (anaphase). But
meiosis differs from mitosis in several aspects of the cell
cycle. Firstly, the G2 phase is dramatically longer and is the
scene of chromosomal pairing and recombination. Secondly,
while mitosis follows (G1 – S – G2 – metaphase – anaphase –
G1)n cycles, meiosis follows a strikingly different pattern
that leads to halving of the chromosomal content, G1 – S –
G2 – metaphase I – anaphase I – interphase – metaphase II –
anaphase II (Fig. 8.4). Two phases of division follow each
other, without an intervening S phase, but just a short inter-
phase without DNA replication.
Precisely how the mitotic machinery is modified for
the purpose of meiosis is still poorly understood. Most of
the knowledge currently available originates from studies
132
carried out in unicellular fungi, Xenopus laevis or mouse
oocyte systems (Marston and Amon 2004). Cyclins and
CDKs form complexes that are essential for progression
through both the mitotic and meiotic cell cycles. High
CDK activity is required for entry and progression into
metaphase and rapid decrease of CDK activity, mediated
by the Anaphase Promoting Complex (APC), is required
for induction of anaphase and exit from the division phases.
The transition from meiosis I to meiosis II requires a fine
balance in cyclin–CDK activity: it must be sufficiently low
to exit meiosis I but must nonetheless be maintained at a
level sufficiently high to suppress DNA replication and
promote entry into meiosis II (Marston and Amon 2004;
Pesin and Orr-Weaver 2008).
While only a few years ago nothing was known about the
control of meiotic progression at the molecular level in
plants this is an exciting time for the field. Recently,
CDKA activity was shown to indeed oscillate throughout
the course of plant meiosis (Bulankova et al. 2010) and
several genes involved in crucial transitions were function-
ally characterized in Arabidopsis (OSD1, TAM, SMG7,
TDM).
Two of these genes, TAM and OSD1 are required for the
transition from meiosis I to meiosis II. Null osd1 and tam
mutants fail to enter the second division in both male and
female meiosis and exit meiosis after anaphase I, leading
to the production of functional 2n gametes (d’Erfurth
et al. 2009, 2010; Wang et al. 2010b). Interestingly, male
meiocytes lacking both OSD1 and CYCA1;2/TAM fail to
enter meiotic division I, producing tetraploid spores directly
after prophase (d’Erfurth et al. 2010). This shows that in
addition to their crucial function in driving the meiosis I to
meiosis II transition, these two genes are involved in the
prophase to metaphase I transition (Fig. 8.4). This suggests
that they both contribute to an activity, most likely mediated
by CDK, which promotes entry into metaphase I and into
metaphase II. The molecular function of OSD1 is currently
unknown, however, by analogy to fission yeast Mes1, it may
inhibit APC activity, thus promoting CDK activity
(d’Erfurth et al. 2009). The cyclin TAM/CYCA1;2, may
directly or indirectly promote the CDK activity controlling
cell cycle progression.
The two other genes, SMG7 and TDM, appear to have
opposite effects on cell cycle progression from the above
genes in the meiotic cycle. In smg7, meiosis arrests at late
anaphase II and cells do not exit from meiosis (Fig. 8.4).
SMG7 is a nonsense-mediated mRNA decay factor sugg-
esting an unappreciated link between the cell cycle machin-
ery and RNA processing (Riehs et al. 2008). Mutants in the
TDM gene have an even more spectacular phenotype: meio-
sis progresses normally up to the end, performing the two
divisions, but then an aberrant third round of division takes
E. Jenczewski et al.
place, without replication (Ross et al. 1997; Glover et al.
1998) (Fig. 8.4). TDM1 has limited similarity with Xe-p9, a
regulatory subunit of CDK. Genetic interaction studies
revealed complex interactions between these genes. tdm is
epistatic to smg7, suggesting that the anaphase II arrest of
smg7 depends (directly or indirectly) on the presence of
TDM (Bulankova et al. 2010). Strikingly, double tam-2/
tdm and tam-2/smg7 mutants exhibit the tdm and smg7
phenotype, respectively (Bulankova et al. 2010). Thus, the
absence of either TDM or SMG7 removes the requirement
for TAM for entry into meiosis II (Fig. 8.4). Remarkably,
while osd1 and tam single mutants have very similar
phenotypes (d’Erfurth et al. 2010), the double osd1/tam
mutant shows the osd1 phenotype with meiotic exit at the
end of meiosis I (Raphaël Mercier et al., unpublished data).
This suggests that OSD1 and TAM act differently on the
progression of meiosis. Altogether, these recent studies
highlight the existence of a very complex network (not yet
well understood!) to regulate the meiotic process.
Two other genes may also affect the meiotic cell cycle,
AtPS1 and JASON (d’Erfurth et al. 2008; Erilova et al.
2009). Both mutants produce diploid spores because of a
defect in spindle orientation at the second division in the
case of Atps1 and through an as yet unclear mechanism in
the case of jas. In both cases it is reasonable to suggest that a
defect in cell cycle/phase identity could generate the
observed phenotype. Interestingly, AtPS1 possesses a PINc
domain that is present in proteins involved in RNA
processing, suggesting that, like SMG7, there is a link
between RNA decay and the meiotic cell cycle.
The identification of a series of genes involved in meiotic
progression and the striking effect of their disruption alone
or in combination, provides a unique opportunity to decipher
the meiotic cell cycle. Study of the meiotic cell cycle
machinery will certainly help to decipher general cell cycle
mechanisms, mirroring the massive contribution of meiosis
research to the understanding of DNA recombination mech-
anisms in somatic cells (Szostak et al. 1983; Paques and
Haber 1999).
Interestingly cell cycle machinery genes, beyond their
function in controlling meiotic progression, appear to have
been recruited to control other key features of meiosis such
as meiotic recombination. Indeed, the SDS gene, which
clearly encodes a cyclin, controls the choice of template
(e.g., homologous versus sister chromatid) during recombi-
nation (De Muyt et al. 2009b) (see above). Similarly, the
wheat Ph1 locus that controls homoeologous recombination
(see above) contains CDK-like genes.
Conclusion
This is an exciting time with an international effort in
progress to better understand the molecular processes
8 Meiosis: Recombination and the Control of Cell Division
involved in meiosis in a large variety of higher
eukaryotes. In plants, thanks to powerful genetic studies
and the development of new efficient molecular tools,
these studies have enjoyed a boom in the last 10 years
with more than 50 genes identified mainly in Arabidopsis
thaliana but also in rice, maize, and wheat. Results
obtained in plants demonstrate that there is both conser-
vation in the underlying molecular mechanisms but also
some specificity compared to other eukaryotic kingdoms.
There is now an opportunity when analysing plants to
decipher the impact of genome structure on the meiotic
process.
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 Mechanisms of Chromosome Rearrangements
9
Martin A. Lysák and Ingo Schubert

Contents 9.1 Introduction

9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
The striking diversity of land plants is associated with
9.2 It Starts With a Break . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
immense genetic variation manifested also by a wide range
9.3 Primary Chromosome Rearrangements . . . . . . . . . . . . . . . . 138 of genome sizes and chromosome numbers. Nuclear genome
9.3.1 Insertions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
size across land plants varies more than 2,300-fold from
9.3.2 Deletions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
9.3.3 Duplications and Deletions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 ~64 Mb (Genlisea aurea; Greilhuber et al. 2006) to
9.3.4 Inversions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 ~150,000 Mb (Paris japonica; Pellicer et al. 2010; see also
9.3.5 Translocations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Leitch and Leitch 2013, this volume). Accordingly, chromo-
9.4 Secondary Chromosome Rearrangements . . . . . . . . . . . . . 141 some numbers can vary from n ¼ 2 in six angiosperm spe-
9.5 Changes of Chromosome Number (Dysploidy) . . . . . . . . 141
cies (Vanzela et al. 1996; Cremonini 2005) to n > 320 in the
9.5.1 Descending Dysploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 angiosperm Sedum suaveolens (Uhl 1978) and n ¼ c. 720 in
9.5.2 Ascending Dysploidy (Centric Fission) . . . . . . . . . . . . . . . . . . . 143 the fern Ophioglossum reticulatum (Khandelwal 1990). This
9.5.3 Bidirectional Dysploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 extensive variation of chromosome numbers among land
9.6 Centromere Rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 plants is driven by two main trends in opposite directions:
9.7 Rearrangements Mediated by Transposable Elements 145 chromosome numbers increase through polyploidy (whole-
genome duplications, WGD) and decrease through structural
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
chromosome rearrangements (descending dysploidy). Geno-
mic and cytogenetic analyses indicate that probably all land
plants have experienced at least one WGD event (Jaillon
et al. 2009; Soltis et al. 2009; Van de Peer et al. 2009; see
also Fawcett et al. 2013, this volume) followed by more or
less extensive karyotype reshuffling towards diploid-like
genomes (e.g., Wolfe 2001; Thomas et al. 2006; Cenci
et al. 2010; Mandáková et al. 2010a). Karyotypic changes
at a given ploidy level are mediated by chromosome
rearrangements such as insertions, duplications, deletions,
inversions and translocations altering the size and morphol-
ogy of chromosomes. Centric fissions and different types
of reciprocal translocations combined with meiotic (mis)
segregation may lead to a reduction or increase of chromo-
some number (descending/ascending dysploidy).
Here we discuss mechanisms which alter size, shape and
number of chromosomes, reviewed earlier by Darlington
(1937), Stebbins (1971), Jones (1998), Levin (2002), Schubert
M.A. Lysák (*) (2007) and Schubert and Lysák (2011). In particular, we
Laboratory of Plant Cytogenomics, CEITEC – Central European
Institute of Technology, Masaryk University, Kamenice 5, CZ-625 00
focus on chromosome rearrangements that have an impact
Brno, Czech Republic on the alteration of chromosome number during evolution of
e-mail: lysak@sci.muni.cz

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 137

DOI 10.1007/978-3-7091-1160-4_9, # Springer-Verlag Wien 2013
138 M.A. Lysák and I. Schubert

land plants and likely play a role in speciation. The emphasis Deletion
is put on chromosome rearrangements detectable by micro-
scopic techniques, whereas small-range insersion/deletion
events (indels), inversions or gene conversion are beyond
the scope of this review. Inversion
a b c d

b c
9.2 It Starts With a Break . . . a c b d

a d
Each chromosome aberration starts with one or more
double-strand break(s) (DSB). DSBs arise either directly
Duplication/deletion
after the exposure to S-phase-independent mutagens (e.g.,
ionizing radiation such as X-rays or gamma rays, chemicals
such as bleomycin or restriction endonucleases), or indi-
rectly when the replication fork meets a repair-mediated
single strand gap. DSBs can be repaired restoring the
original DNA structure, whereas a mis-repair of two or Interchromosomal translocation
more simultaneous DSBs results in a chromosome rear-
rangement. DSBs are repaired by homologous recombina- a b c d a b h i j
tion (HR), using sequences homologous to the broken e f g h i j e f g c d
strands as a template for DNA synthesis and operating
mainly in S and G2-phase or by non-homologous end Fig. 9.1 Primary chromosome rearrangements mediated by non-
joining (NHEJ, also known as illegitimate recombination), allelic homologous recombination (NAHR). Grey arrows represent
which can join broken ends directly without or with just a homologous (repetitive) sequences (redrawn and modified after
Griffiths et al. (2007))
few base pairs of homology. HR is a mostly accurate repair
mechanism, whereas NHEJ is an error-prone process usu-
ally associated with microdeletions and insertions (filler
DNA) prior to ligation of broken ends. HR can also occur
9.3 Primary Chromosome Rearrangements
between ectopic homologous sequences. Non-allelic
9.3.1 Insertions
(ectopic) homologous recombination (NAHR) may occur
within one double helix, between sister chromatids, or
DSB repair can be accompanied by insertion of endogenous
between chromatids of homologous or non-homologous
or alien sequences. A possible mechanism of insertion can
chromosomes. Recombination using different templates
be recombination with extrachromosomal circular DNA
has different outcomes as shown in Fig. 9.1 (see also
(Cohen et al. 2008; Navrátilová et al. 2008) (Fig. 9.2a).
Gaut et al. 2007; Gu et al. 2008). Intrastrand recombination
between direct repeats results in deletion (via “pop-out” of
a small circular chromosome segment). If the repeats have
the opposite orientation, NAHR yields an inversion of 9.3.2 Deletions
the region between the breakpoints. HR between ectopic
homologous sequences of sister chromatids in direct orien- Deletions can be terminal or interstitial. After loss of a
tation produces chromatids and chromosomes with dupli- terminal chromosome segment, the broken end has to be
cation and deletion, respectively (unequal sister chromatid healed and stabilized by de novo telomere synthesis or by
exchange). This process is analogous to unequal crossover telomere capture (acquiring telomeric sequence from a sister
at meiosis. NAHR between repeats on homologous or non- chromatid, a homologous or heterologous chromosome via a
homologous chromosomes results in reciprocal chromo- conversion-like mechanism; Yu and Graf 2010), otherwise
some translocations. the broken chromosome ends enter a fusion-breakage cycle
Both repair mechanisms, HR and NHEJ, are active in or the harbouring cell dies. Interstitial deletions require two
somatic cells, while allelic meiotic recombination is exclu- breaks and arise e.g., via NHEJ between the most proximal
sively based on HR, processing Spo11-induced DSBs. In and the most distal break end (Fig. 9.2b). During meiotic
plants, rearrangements occurring in somatic tissues (shoot pairing in an interstitial-deletion heterozygote, the wild-type
meristems) can be transmitted to the next generation. chromosome loops out the non-deleted region.
9 Mechanisms of Chromosome Rearrangements
a b
d e
g h
Alternate
Fig. 9.2 Primary chromosome rearrangements. (a) Insertion of circular
extrachromosomal DNA. (b) Interstitial deletion. (c) Duplication and
deletion caused by unequal meiotic crossover between homologous
chromosomes. (d) Paracentric inversion. (e) Pericentric inversion.
(f) Intrachromosomal translocation resulting in a ring chromosome.
(g) Symmetrical reciprocal translocation. (h) Asymmetric reciprocal
translocation yielding a dicentric chromosome and an acentric fragment;
9.3.3 Duplications and Deletions
Large-scale (>10 kb) duplications usually coupled with
simultaneous deletion events may originate through multiple
mechanisms. A duplicated segment is either placed adjacent
to the original sequence (tandem duplication) or elsewhere
139
c
f
or
Adj 1
Adj 2
the dicentric is destroyed by a rupture of the anaphase bridge when there
is an intercentromeric sister chromatid twist (far right). (i) Meiosis I in a
symmetrical translocation heterozygote. The alternate segregation will
result in balanced gametes with a “normal” and translocation chromo-
some complement, respectively. Adjacent (Adj) segregation yields
unbalanced gametes with duplications and deletions, via adjacent segre-
gation of non-homologous (Adj-1) or homologous centromeres (Adj-2)
in the complement (insertional duplication). During meiotic
prophase of a tandem-duplication heterozygote, the unpaired
duplicated region forms a loop. Erroneous repair of two
DSBs via unequal sister chromatid exchange or via unequal
meiotic crossover (CO) between two not well aligned homol-
ogous chromatids may result in duplication and deletion
140
(Fig. 9.2c). NHEJ frequently accompanied by exonucleo-
lytic digestion at DSB ends or by insertional filling of
sequences into the break may cause deletions and duplications
at the submicroscopic level. Tandem duplications generate
additional substrates for ectopic homologous recombination
(Fig. 9.1); thus increasing the probability of further rearran-
gements caused by this mechanism.
9.3.4 Inversions
Inversions are intra-chromosomal rearrangements that alter the
collinearity due to the opposite orientation of a chromosome
segment, without changing the chromosome size. Inversions
are induced by two breaks followed by the reunion of the
internal fragment with the ‘wrong’ break ends. Traditionally
paracentric and pericentric inversions have been recognized.
A paracentric inversion does not include the centromere
(Fig. 9.2d) and thus does not change the chromosome mor-
phology, if no breakpoints at different distances on either side
of a nucleolus organizing region (NOR) are involved.
A pericentric inversion, including the respective centromere,
may result in a changed arm ratio, if the breakpoints on either
side have a different distance to the centromere (Fig. 9.2e).
Ectopic homologous recombination (see Sect. 9.2), for
instance between two transposon copies (Delprat et al.
2009), seems to be a mechanism frequently yielding chro-
mosome inversions. Two DSBs (Runcie and Noor 2009) or
the coincidence of two pairs of staggered single-strand
breaks eventually repaired by NHEJ (Ranz et al. 2007)
may lead to inversions without requiring homologous motifs
between the inversion breakpoints.
During meiotic pairing in inversion heterozygotes the
inverted region forms an ‘inversion loop’ with the non-
inverted homologous regions (including the centromere in
heterozygotes for pericentric inversion). If no CO occurs
within the inversion loop, 50% of the gametes will carry the
inversion. However a CO within the inversion loop of a
heterozygote for a pericentric inversion will result in normal
(1), inversion (1) and duplication/deletion (2) chromatid types.
In a heterozygote for a paracentric inversion, a CO within the
inversion loop connects homologous chromosomes to form a
dicentric and an acentric fragment (the latter lost as a micro-
nucleus). At anaphase I, the dicentric bridge is randomly
broken yielding two chromatids with terminal deletions. One
normal and one inversion chromatid are also produced.
9.3.5 Translocations
Translocations are induced by two DSBs resulting in four
DNA ends. An erroneous repair by ligation of the ends of
different breaks generates a reciprocal translocation. In this
M.A. Lysák and I. Schubert
case, DSBs are mis-repaired by NHEJ not requiring sequence
homology at the breakpoints, but DSBs can also be joined
through HR between ectopic repeats (see Sect. 9.2). NAHR-
mediated translocations are facilitated by the abundance of
repetitive elements which are common in plants with large
genome sizes. An intrachromosomal reciprocal translocation
with a break in both arms yields a ring chromosome and a
terminal acentric fragment (Fig. 9.2f). The ring chromosome
(particularly if very small) can be stably inherited for some
time (Murata et al. 2008) but usually both translocation
products are unstable and lost. More common are interchro-
mosomal reciprocal translocations (interchanges) which may
involve homologous or heterologous chromosomes and can
be either symmetric and yield monocentric products
(Fig. 9.2g), or asymmetric, resulting in a dicentric and an
acentric product (Fig. 9.2h). Dicentric products of asymmet-
ric translocations are destroyed by rupture of anaphase
bridges if inter-centromeric sister chromatid twists occur
and the two centromeres are pulled to the opposite poles.
Dicentrics might be stable if both centromeres are in close
vicinity and are pulled to one pole (Fig. 9.2h). Inactivation
and/or loss of one centromere can turn the dicentric chromo-
some into a stable monocentric (see Sect. 9.6).
During the first meiotic division in a translocation hetero-
zygote, normal and translocated chromosomes pair to form
a cruciform configuration (Fig. 9.2i). Depending on the
number and localization of chiasmata, different chromosome
configurations (e.g., a ring or chain of chromosomes) are
observed in diplotene and diakinesis. Due to the independent
assortment of chromosomes, two types of segregation can
occur (Fig. 9.2i). During alternate segregation, the transloca-
tion chromosomes are pulled to one pole and “normal”
chromosomes to the other pole. This segregation eventually
yields balanced gametes with two normal and two transloca-
tion chromatids. In so-called adjacent segregation, either both
“adjacent” non-homologous or homologous centromeres pass
to the same pole. Both types of adjacent segregation result in
unbalanced gametes with duplications and deletions. Such
gametes are usually not viable, but may be tolerated in
polyploids. If so, the progeny genomes may be erroneously
considered as bearing a ‘non-reciprocal translocation’ (in
polyploids also called homoeologous non-reciprocal transpo-
sition or translocation; Udall et al. 2005; Gaeta et al. 2007;
Nicolas et al. 2007) which have not yet been proven experi-
mentally. Translocation events involving unequally sized
chromosome regions may also be erroneously interpreted as
a “non-reciprocal” translocation. In this case, besides a large
translocation product, very small centric or acentric fragments
are also generated (see Sect. 9.5.1). As these products may be
either undetectable by routine microscopic techniques or
eliminated during subsequent mitotic (acentric fragments) or
meiotic divisions (centric fragments), the translocation event
might appear as non-reciprocal within the progenies.
9 Mechanisms of Chromosome Rearrangements
a b
wt
Dp Dp
Dp
Dp
wt
Fig. 9.3 Secondary chromosome rearrangements. (a) Two trans-
locations (① and ②) involving three chromosomes of the wild-type
(wt) karyotype lead to hexavalents during meiosis in double heterozy-
gous individuals. Cross over between homologous chromosome regions
flanked by non-homologous regions generates a novel karyotype and
re-establishes the wt complement. (b) Origin of new karyotypes through
primary chromosome rearrangements and meiotic recombination. One
chromosome is involved in an inversion in one individual (①) and in a
translocation in another individual (②). In the resulting quadrivalent,
crossover within homologous chromosome regions flanked by non-
homologous regions generates two new karyotypes with complementary
duplication (Dp) and deletions, respectively
Translocated regions might be lost or duplicated due to
CO between rearranged chromosomes (Fig. 9.3b), or due to
meiotic mis-segregation in translocation heterozygotes
(Fig. 9.4g) yielding unbalanced karyotypes.
Short range gene conversion should not to be considered
as non-reciprocal translocation.
9.4 Secondary Chromosome
Rearrangements
Structural chromosome alterations can additionally arise as
secondary chromosome rearrangements (for review see
Schubert 2007). Such rearrangements may arise in
organisms doubly heterozygous for two primary rearran-
gements (translocations and/or inversions), if one chromo-
some is involved in both of them (Fig. 9.3). Meiotic CO
between homologous regions of rearranged chromosomes,
which differ in the regions distal to the CO, leads to gametes
with a new karyotype and to complementary gametes
displaying a re-established wild type chromosome comple-
ment (Fig. 9.3a). Depending on the type of primary
141
rearrangements involved, unbalanced gametes may also
arise, harbouring duplications as well as deletions (Fig. 9.3b;
Schubert et al. 1988).
9.5 Changes of Chromosome Number
(Dysploidy)
Descending and ascending dysploidy is a karyotypic alter-
ation towards a lower or higher chromosome number.
Translocations play a prominent role in decreasing chromo-
some numbers. The increase of chromosome number, apart
from polyploidization, is mediated by chromosome fission
(dissociation). The evolutionary success of novel karyotypes
depends on the viability of gametes and on whether or not
homozygosity is possible under natural selection.
9.5.1 Descending Dysploidy
A Robertsonian (Rb) translocation or centric fusion refers to
a translocation between two telocentric or acrocentric
chromosomes. This translocation event results in a meta
(di)centric ‘fusion’ chromosome (Sullivan et al. 1996) and
a small acentric fragment (Fig. 9.4a), or in two monocentric
products of unequal size (Fig. 9.4c). If the minichromosome
is eliminated during subsequent cell divisions, the Rb event
may lead to a heritable decrease in chromosome number
(n1). Typically, Rb breakpoints are localized at the centric
ends of the short arms of telo- or acrocentric chromosomes
[i.e., (sub)telomere in telocentrics]. As a consequence, Rb
translocations reduce chromosome number but maintain the
number of major chromosome arms (the nombre
fondamental, NF). The ribosomal DNA (rDNA) of terminal
NORs was thought to mediate Rb translocations. Often
breakpoints were found proximal to NORs on acrocentric
chromosomes and the NORs were deleted (Page et al. 1996).
Several human Rb metacentrics have originated through
variably positioned breakpoints within the short arms and
less frequently at the pericentromeres of the two acrocentric
chromosomes involved (Page et al. 1996; Sullivan et al.
1996). Hence, 90% of Rb chromosomes in humans are
dicentric (Page et al. 1996). Although an exact mechanism
of Rb fusion remains to be elucidated, the abundance of
satellite DNA arrays on short arms of acrocentrics and at
centromeres of Rb chromosomes suggests NAHR based on
ectopic homologous sequences as a mechanism (Page et al.
1996; Sullivan et al. 1996). Rb fusions in plant species can
also result in dicentric chromosomes as shown for the meta-
centric chromosome 1 of Vicia faba (Schubert 1992). In the
crucifers (Brassicaceae) (and likely in other groups),
Rb translocations are frequently preceded by a pericentric
inversion in one of the chromosomes, rendering a (sub)
142 M.A. Lysák and I. Schubert

a
b

d
c

g
e

Dp
[ Dp

Fig. 9.4 Chromosome rearrangements mediating descending and chromosome can be prone to fission which yields two telocentric
ascending dysploidy. (a) Robertsonian translocation between two telo-/ chromosomes in the presence of telomerase (stars) stabilized by de novo
acrocentric chromosomes with breakpoints in short arms results in a meta added telomeres. (b) Centric fission in a monocentric chromosome. Cen-
(di)centric chromosome and an acentric fragment. The dicentric tric ends of fission products are “healed” by telomerase (stars).
9 Mechanisms of Chromosome Rearrangements
metacentric chromosome telo- or acrocentric. This rear-
rangement allows for a Rb event resulting in a large “fusion”
chromosome and a minichromosome prone to loss (Fig. 9.4c;
Lysák et al. 2006). For chromosome number reduction in
some cruciferous species, centromere inactivation and/or
loss has been envisaged to follow asymmetric, end-to-end
translocation between two metacentrics (Fig. 9.4d; Mandáková
et al. 2010a, b).
Based on genomic data, insertional or nested chromosome
fusion (NCF) has been proposed as a prominent mechanism
of descending dysploidy in grasses (Luo et al. 2009; Salse
et al. 2009; Thiel et al. 2009; Abrouk et al. 2010; Interna-
tional Brachypodium Initiative 2010; Murat et al. 2010), but
it is considered to occur less frequently in cruciferous taxa
(Mandáková et al. 2010b). In NCF, one chromosome is
inserted between the chromosome arms of another (recipi-
ent) chromosome. Although not yet proven experimentally, a
NCF can most easily be interpreted as the outcome of a
multiple breakage and translocation event between two
non-homologous chromosomes. Simultaneous breaks at the
ends of both arms of the nested chromosome and one around
the centromere of the recipient chromosome are followed by
a mis-repair of the chromosome breaks (Fig. 9.4e). Disrup-
tion of the recipient centromere by a break followed by the
inactivation of its parts is a plausible mechanism to avoid an
unstable di- or tricentric product. Alternatively, if the recipi-
ent chromosome has breaks on either side of its centromere, a
symmetrical translocation may yield two monocentric
products: the ‘fusion’ chromosome and a minichromosome
comprising the centromere of the recipient chromosome and
the telomeres of the inserted chromosome. The small product
without essential genes is prone to loss during meiosis
(Fig. 9.4f). Theoretically, the two arms of the recipient chro-
mosome could have been translocated to the nested chromo-
some arm ends via two subsequent events. However, it is
hard to explain why the second event in so many cases again
involved the same two chromosomes (Luo et al. 2009; Thiel
et al. 2009; International Brachypodium Initiative 2010).
A simultaneous translocation of broken donor chromosome
arms to different recipient chromosomes would bear a higher
risk of unbalanced gametes resulting from segregation of
meiotic multivalents.
ä
Fig. 9.4 (Continued) (c) Translocation between two chromosomes
with breakpoints in the pericentromere of the long arm of one chromo-
some and in the telomeric end of the short arm of another chromosome.
This translocation yields a large monocentric “fusion” chromosome
and a meiotically unstable minichromosome. Involved chromosomes
can become telo-/acrocentric via a preceding pericentromeric inversion
(far left). (d) End-to-end reciprocal translocation between two (sub)
metacentric chromosomes followed by inactivation/loss of one centro-
mere. (e, f) Proposed mechanisms of nested chromosome “fusion”
through three (e) or four (f) simultaneous double strand breaks
(DSBs) in two non-homologous chromosomes. In (e) the centromere
143
9.5.2 Ascending Dysploidy (Centric Fission)
Centric fission, i.e., dissociation of two arms of usually a
metacentric chromosome increasing the chromosome num-
ber (n þ 1), is considered to counteract Robertsonian fusion
(Sect. 9.5.1). Centric fissions are a favoured explanation for
the origin of telocentric chromosomes. However, to decide
whether acro-/telocentric chromosomes result from fission
or from a whole-arm pericentric inversion in a (sub)meta-
centric has been difficult without recently established chro-
mosome painting or comparative genomics. The mechanism
of centric fissions, more often observed in animals than
plants, remains controversial. Each of the two telocentric
fission products should possess a functional centromere and
telomere sequences capping the centric ends. “Chromosome
healing” by de novo synthesis of telomeres or telomere
capture (Fig. 9.4a, b) has been postulated. Moreover, inter-
stitial telomere(like) repeats identified in centromeric
regions of some species may serve as telomere seeds in
fission telocentrics. In a fission heterozygote of the plant
Hypochaeris radicata, both fission products were stabilized
at centric ends by transposition of 45S rDNA from a NOR of
another chromosome (Hall and Parker 1995). Apparently,
rDNA is a potential substrate for telomere synthesis by
telomerase at chromosome breakpoints through 2–4-nucleotide
target motifs within the rDNA sequence (Tsujimoto et al.
1999). The transposition and amplification of rDNA is
concordant with the post-fission extension of constitutive
heterochromatin transforming fission telocentrics into
acrocentrics as proposed by Imai (1991).
A transversal division of the centromere into two func-
tional parts represents a challenging problem (Fig. 9.4a, b).
A stable dicentric Robertsonian translocation chromosome
provides functional centromeres when a break occurs
between the two centromeric regions (Fig. 9.4a and Schubert
et al. 1995). Fission events in monocentrics (Fig. 9.4b) were
explained by at least two mechanisms. Perry et al. (2005)
carried out detailed molecular analyses of centric fissions in
human chromosomes and found evidence for simple fission
events as well as for centromere duplication prior to fission.
In the latter case duplication of (peri)centromeric sequences
is predisposing the centromere to breakage. An alternative
of the recipient chromosome is disrupted by a single DSB, whereas in
(f) it is lost as a second, meiotically unstable translocation product.
(g) Simultaneously ascending and descending dysploidy in double
heterozygous individuals. Crossing of two karyotypes heterozygous
for two different translocations involving three chromosomes may
result in mis-segregation from meiotic multivalents. The two metacen-
tric translocation chromosomes segregate to one pole (n1) and the
four acrocentric chromosomes to the other (n þ 1). The hypoploid
gametes harbour small deletions, whereas hyperploid gametes carry
the corresponding duplications (Dp)
144
a b
Fig. 9.5 Alternative mechanisms of centromere repositioning (CR).
(a) CR through the emergence of a neocentromere and inactivation of
the original centromere. The old pericentromere (grey) decays and new
pericentromeric sequences accumulate at the neocentromere. (b) CR via
subsequent peri- and paracentric inversions. The paracentric inversion
restores the original collinearity along the chromosome, mimicking the
mechanism is based on coupling fission events with the
appearance of neocentromeres (Nasuda et al. 2005). The
origin of human acrocentric chromosomes 14 and 15 from
an ancestral metacentric chromosome was interpreted
assuming (1) a noncentromeric break within the ancestral
metacentric, (2) inactivation of the ancestral centromere and
the appearance of a neocentromere on HSA15, and (3)
formation of a neocentromere on HSA14 (Fig. 9.1 in
Ventura et al. 2003).
9.5.3 Bidirectional Dysploidy
The occurrence of two translocations between three
chromosomes (one of the three involved in both
translocations) with all breakpoints close to the centromeres
may result in simultaneous ascending and descending
dysploidy (Fig. 9.4g). In individuals double-heterozygous for
both translocations, a hexavalent is formed during meiotic
chromosome pairing. At a low frequency, the two transloca-
tion metacentrics of the hexavalent segregate to one pole and
the four acrocentrics to the other. Consequently, gametes
containing the acrocentric translocation chromosomes possess
one chromosome more than the parental lines and a duplica-
tion of at least one centromere plus two terminal regions, while
gametes with the metacentric translocation chromosomes have
one chromosome less and the corresponding deletions. When
gametes of the same dysploidy fuse, the chromosome number
can increase or decrease simultaneously in a homozygous
fashion, provided the accompanying duplications and
deletions can be tolerated. This dysploidy mechanism has
been experimentally proven in Vicia faba (Schubert and
Rieger 1985). Similar mechanisms have been suggested
already by Stebbins (1971). The increase in chromosome
number from n ¼ 10 to n ¼ 12 postulated for a common
(tetraploid) ancestor of cereals (Salse et al. 2008) could be
explained in the same way. After whole genome duplication
(¼ polyploidy) events, in the course of genome diploidization,
M.A. Lysák and I. Schubert
c
mechanism described under (a). Pericentromeric arrays at the original
centromere decay and are re-established at the new centromeric position.
(c) CR mediated by the deletion of a centromere through centromere-
flanking DSBs and its re-insertion into another DSB on the same
chromosome
a broad range of chromosome rearrangements can be tolerated
(Mandáková et al. 2010a, b), and dysploidy, including
deletions or duplications, is likely to become fixed. However
it can be difficult to interpret these events subsequently as
seen, for example, in the descending dysploidy leading to the
extant maize genome (Wei et al. 2007; Salse et al. 2008, 2009).
The more distant from the centromere the breakpoints in the
translocation chromosomes of the double heterozygous
individuals are, the larger are the duplicated (and the
corresponding deleted) regions in the dysploid progeny
karyotypes. The resulting segmental duplications or deletions
may therefore give the impression of ‘non-reciprocal
translocations’.
9.6 Centromere Rearrangements
The classical theory of chromosome rearrangements consid-
ered centromeres as evolutionary stable chromosome
structures, which may be lost or gained only by translocation
or centric fission events. Consequently, until recently
(Voullaire et al. 1993) positional changes of centromere
position as well as emergence or loss of centromeres have
been explained based on primary chromosome rearran-
gements. Today circumstantial evidence suggests the possi-
bility of so-called centromere repositioning (CR), assuming
centromere inactivation at one site and the emergence of a
new centromere at another site. These events might occur
without distortion of the marker order along the chromo-
some (Fig. 9.5). Such a centromere shift may change the arm
ratio and thus the shape of the corresponding chromosome.
CR events are claimed for vertebrates (Ventura et al. 2007;
Piras et al. 2010) and have been presumed for homoeologous
chromosomes of cucumber and melon (Han Y. et al. 2009).
It remains however unclear how during the transition phase
of CR (old centromere no longer active and neocentromere
just emerging) the chromosome resolves the problem of
having either no active centromere or two centromeres.
9 Mechanisms of Chromosome Rearrangements
Although some dicentric human chromosomes were shown
to be stable over several cell generations (Stimpson and
Sullivan 2010), more experimental data are needed to further
elucidate this process. Apart from a presumably epigenetic
mechanism of CR (Fig. 9.5a), subsequent peri- and para-
centric inversions may alter centromere position and restore
collinearity (Fig. 9.5b). Alternatively, CR can be mediated
by the deletion of a (peri)centromere and its re-insertion into
another region of the same chromosome (Fig. 9.5c).
Centromere inactivation is not only postulated to occur
during a shift of centromeres but may also play a role in
stabilizing dicentric chromosomes. The inactivation of one
centromere on a dicentric maize B chromosome was caused
by the loss of epigenetic centromere markers such as
CENH3, CENP-C and H3S10P. The inactive centromere
could be reactivated when separated from the active centro-
mere by intrachromosomal recombination (Han F. et al.
2009). This is compelling evidence that epigenetically
inactivated centromeres can be reactivated. Centromere
inactivation has also been observed in dicentric human
chromosomes (e.g., Stimpson and Sullivan 2010) and it has
been suggested to stabilize primary dicentric chromosomes
resulting from telomere-to-telomere translocations in
some cruciferous species (Fig. 9.4d; Mandáková et al.
2010a, b). Mechanisms of centromere inactivation are not
well understood.
As centromere formation and function seem to be deter-
mined epigenetically without specific sequence requirements,
a neocentromere could also emerge in acentric fragments and
thus increase chromosome number. In plants, the emergence
of a new centromere was shown to occur on fragments of
barley chromosomes (Nasuda et al. 2005). As shown for plants
(Han F. et al. 2009) and vertebrates (Rocchi et al. 2009),
some neocentromeres occur at sites of inactivated ancestral
centromeres.
9.7 Rearrangements Mediated
by Transposable Elements
Barbara McClintock was the first to recognize that the
mobility of transposable elements could cause chromosome
rearrangements such as translocations, inversions, dupli-
cations and deletions (e.g., McClintock 1948). Indeed there
is now cytological evidence showing how the origin of
translocations and inversions in maize is due to the alterna-
tive transposition of Ac elements (Pulletikurti et al. 2009).
Whereas conventional transposition is just moving a single
element to another genomic position, alternative trans-
position events involving termini of two elements can
cause chromosome breakage and major chromosome
rearrangements (Weil 2009; Zhang et al. 2009). Models to
explain different chromosome rearrangements resulting from
145
alternative transposition of maize Ac/Ds transposons are
available at http://jzhang.public.iastate.edu/transposition.
html, and as supplemental materials online in Zhang et al.
(2009).
Some transposable elements show the capacity to capture
and move other genomic sequences. In maize, for example,
Helitron transposons replicating through a rolling-circle
mechanism frequently capture neighbouring sequences
such as gene fragments (Yang and Bennetzen 2009). Trans-
position of larger chunks of chromosomal DNA has been
suggested for repeated arrays of the large ribosomal RNA
genes (rDNA) discernible on chromosomes as NORs. Since
Schubert and Wobus (1985) showed the mobility of NORs
(“jumping NORs”), even in cells of the same individual,
intraspecific variation in number, position and extension of
rDNA loci has been revealed in a wide range of plant species
(e.g., in the tribe Triticeae, Dubcovsky and Dvořák 1995).
As the observed variation in number and position of NORs
between different individuals or populations is often not
associated with collinearity distortion (Datson and Murray
2006), the NOR mobility is explained by transpositional
‘hitch hiking’ together with flanking transposable elements.
Indeed, Raskina et al. (2004, 2008) showed by fibre fluores-
cence in situ hybridization that transposable elements can be
interspersed with 5S and 45S rDNA repeats in Aegilops and
Triticum species suggesting that the mobility of transposable
elements is responsible for the extensive intraspecific varia-
tion in the number and localization of rDNA loci in the
analysed grass species.
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 Biology and Evolution of B Chromosomes
10
Andreas Houben, Ali Mohammad Banaei-Moghaddam,
and Sonja Klemme

Contents 10.1 Introduction

10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Supernumerary or B chromosomes (Bs) are dispensable
10.2 Structure and DNA Composition of B Chromosomes 150
10.2.1 Centromere Organization of B Chromosomes . . . . . . . . . . 150 components of the genomes of numerous plant, fungi, and
10.2.2 B Chromosome-Located Genes . . . . . . . . . . . . . . . . . . . . . . . . . . 151 animal species. They do not pair with any of the standard A
10.2.3 DNA Composition of Rye B Chromosomes . . . . . . . . . . . . . 151 chromosomes (As) at meiosis, and have irregular modes of
10.2.4 Chromatin Composition of B Chromosomes . . . . . . . . . . . . 152 inheritance. As they are dispensable for normal growth, Bs
10.3 Effects and Transcripts Associated With B were considered non-functional and without any essential
Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 genes. As a result, B chromosomes follow their own species-
10.3.1 Mitotic and Meiotic Segregation Behaviour of B
Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
specific evolutionary pathways. Because most Bs do not con-
10.3.2 Diverse Drive Mechanisms of B Chromosomes . . . . . . . . 156 fer any advantages on the organisms that harbour them, they
may be thought of as parasitic elements that persist in
10.4 Evolution of B Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . 158
populations by making use of the cellular machinery required
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 for the inheritance and maintenance of A chromosomes. In low
numbers Bs show little or no impact on the hosts. However,
increased numbers of Bs cause phenotypic differences and
reduced fertility, reviewed in Jones and Rees (1982), Jones
(1991, 1995), Bougourd and Jones (1997), Camacho et al.
(2000), Camacho (2004, 2005), Jones and Houben (2003),
Jenkins and Jones (2004), and Jones et al. (2008a, b).
B chromosomes are a major source of intraspecific varia-
tion in nuclear DNA amounts in numerous species of plants
(Jones et al. 2008b). The distribution of Bs among different
groups of angiosperms is not random. Among flowering
plants they are more likely to occur in outcrossing than inbred
species, and their presence is also positively correlated with
genome size and negatively with chromosome number. They
are not found any more frequently in polyploids than in
diploids (Palestis et al. 2004; Levin et al. 2005). In many
plants different morphological types of Bs exist within a
single species. The relationship between genome size and B
frequency may be explained on the grounds that species with
large genomes can tolerate supernumerary chromosomes
more readily (Puertas 2002) or that the greater amount of
noncoding DNA, which is what largely constitutes large
A. Houben (*) genomes, is itself a trigger for B formation (Levin et al.
Leipnitz-Institute of Plant Genetics and Crop Plant Research (IPK),
Chromosome Structure and Function Laboratory, Corrensstrasse 3,
2005). According to a survey of 23,652 angiosperm species,
06466 Gatersleben, Germany about 8% of monocots and 3% of eudicots have Bs, and of
e-mail: houben@ipk-gatersleben.de

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 149

DOI 10.1007/978-3-7091-1160-4_10, # Springer-Verlag Wien 2013
150
these by far the largest number of species belong to the
Poaceae and Asteraceae. However, because these families
are also highly speciose, several other families have a higher
proportion of species with Bs. Among orders, the two B
chromosome ‘hot spots’ are the Liliales and Commelinales
(Palestis et al. 2004; Levin et al. 2005).
10.2 Structure and DNA Composition
of B Chromosomes
The size of B chromosomes differs from species to species.
Generally Bs are smaller than As, however Bs bigger than
As have been detected in some species, e.g., the cyprinid fish
Alburnus alburnus (Ziegler et al. 2003) and the neotropical
fish Astyanax scabripinnis paranae (Maistro et al. 1992). In
some species even different types of B chromosomes exist,
for example in the plant Brachycome dichromosomatica
large Bs and micro Bs can be observed (Carter and Smith-
White 1972). The marsupial frog Gastrotheca espeletia
possesses three types of Bs which differ among themselves
in size and morphology (Schmid et al. 2002).
Generally, B chromosomes have a similar overall DNA
composition to the respective As of the host species
(Camacho et al. 2000). Early investigations into the DNA
composition of Bs were based on gradient density centrifu-
gation and renaturation kinetics. In rye, these experiments
showed that the ratio and heterogeneity of repeats in Bs did
not differ from As, although a slight increase in cytosine and
guanine content was observed in the DNA from B-carrier
plants (Rimpau and Flavell 1975; Timmis et al. 1975).
Buoyant densities of DNA from maize (Zea mays) plants
with and without Bs were found to be similar (Chilton and
Mccarthy 1973) and the same results were reported in the
mealy bug Pseudococcus obscurus (Klein and Eckhardt
1976) and the grasshopper Myrmeleotettix maculatus (Gib-
son and Hewitt 1970). Later, Amos and Dover (1981)
introduced the use of restriction endonuclease digestion of
genomic satellite DNA to characterize Bs. However, only in
recent years have techniques like chromosome microdissec-
tion (Houben et al. 2001a) and flow-sorting (Kubalakova
et al. 2003; Bartos et al. 2008) evolved enough to allow for
direct isolation of B chromosome-derived DNA. In the
future, efficient and less costly sequencing tools should
allow the analysis of megabase-long DNA fragments
derived from A and B chromosomes. Comparative sequence
analysis will then significantly improve our knowledge on
the origin of Bs, and hence of the evolution of genomes.
Many Bs contain large amounts of repetitive DNA,
especially mobile elements, although strong amplification
of tandem repeats and satellite DNA has also been
observed (Langdon et al. 2000; Dhar et al. 2002). One
such example is the micro B of Brachycome
A. Houben et al.
dichromosomatica which is mainly composed of tandem
repeats (Houben et al. 2001b). The large amounts of repet-
itive elements might facilitate the strong heterochromati-
nization observed in some Bs. Since Bs are cut off from
recombination, a greater proportion of mobile elements
will accumulate compared with the As, and because Bs
are under relaxed selective pressure, they present a kind of
safe spot for mobile elements. Nevertheless, as Bs follow a
separate path of evolution, the composition of their repeti-
tive fraction is expected to differ from that of the As,
depending on the age of the Bs.
Molecular studies have shown that in many species the Bs
contain sequences that have originated from one or several
A chromosomes (Houben et al. 2001b; Page et al. 2001;
Cheng and Lin 2003; Bugrov et al. 2007). Mostly these are
non-coding repetitive sequences or mobile elements like in
the plant Crepis capillaris (Jamilena et al. 1994, 1995),
the fish Prochilodus lineatus (de Jesus et al. 2003), the
fungus Nectria haematococca (Enkerli et al. 1997), and the
insect Drosophila subsilvestris (Gutknecht et al. 1995). In
maize, sequence analysis revealed many highly repetitive
sequences, including transposons which were present on
both A and B chromosomes, but enriched on Bs (Alfenito
and Birchler 1993; Stark et al. 1996; Theuri et al. 2005;
Lamb et al. 2007a). Only a few sequences are considered
B-specific though they are also present on As in minor
traces. For example: the Bd49 tandem repeat in Brachycome
dichromosomatica (Franks et al. 1996) or the B-specific
retroelement in Alburnus alburnus (Schmid et al. 2006). In
rye (Secale cereale) two B-specific sequences, D1100
(Sandery et al. 1990) and E3900 (Blunden et al. 1993),
have been identified. Both are located at the end of the
long arm of this B-type (Wilkes et al. 1995) and reside in
the same region that is supposed to contain one or more
genes controlling the directed non-disjunction of the rye
Bs. More data on characterization of the DNA composition
of Bs are given in Jones and Rees (1982) and Camacho (2005).
10.2.1 Centromere Organization
of B Chromosomes
An understanding of the structure and regulation of A and B
centromeres is a prerequisite for a better understanding of
the unique segregation behaviour of Bs. The B-specific
repeat ZmBs has been used to characterise extensively the
centromeres of maize Bs (Alfenito and Birchler 1993;
Kaszas and Birchler 1996, 1998; Kaszas et al. 2002),
which are amongst the best-characterized plant centromeres.
These studies have shown that the centromeres of maize
Bs contain several megabases of a B chromosome-specific
repeat (ZmBs), a 156-bp satellite repeat (CentC), and
centromere-specific retrotransposons (CRM elements).
10 Biology and Evolution of B Chromosomes
However, only a small fraction of the ZmBs repeats interacts
with the kinetochore protein CENH3 (the histone H3 variant
specific to functional centromeres and also called CENPA).
CentC, which marks the CENH3-associated chromatin in
maize A centromeres, is restricted to a 700-kb domain within
the larger context of the ZmBs repeats (Jin et al. 2005).
Clearly, centromere specification must have an epigenetic
component as dicentric A-B translocation chromosomes are
characterized by the stable inheritance of an inactive state of
one of the centromeres over several generations (Han et al.
2006).
A comparison of maize A and B chromosomes seems to
show that Bs are enriched with DNA elements that are
normally found at or near A centromeres (Lamb et al.
2005). A similar tendency has been described for the rye B,
which is characterized by a likely higher copy number of
the rye retrotransposon-like centromeric repeat pAWRC.1
(Wilkes et al. 1995). However, in contrast to maize Bs
(Lamb et al. 2005; Jin et al. 2008), the rye kinetochore
protein CENH3 is present in equal amounts on both As and
Bs (Houben unpublished).
The centromeric region of B. dichromosomatica standard
Bs (of cytodeme A1, A2, and A4) is enriched with a
B-specific tandem repeat (Bd49) that is not microscopically
detectable on A chromosomes (Franks et al. 1996; Leach et al.
1995). Initially the predominantly centromeric location of the
Bd49 repeat suggested a possible role for this sequence in the
drive process, but a noncentromeric Bd49 location in B.
dichromosomatica cytodeme A3 and differences in signal
sizes among all the Bs of different cytodemes do not support
this assumption (Houben et al. 1999).
10.2.2 B Chromosome-Located Genes
Single or low copy genes are rarely found on Bs. This could
be due to a problem of finding them between the massively
amplified mobile elements. However, many Bs have been
suggested to contain active genes due to visible phenotypic
effects on the host (Camacho 2005). In maize, several
sequences have been isolated that are homologous to non-
coding regions of genes located on the As (Cheng and Lin
2003). Recently, histone genes have been found on the Bs of
the migratory locust Locusta migratoria, although these genes
appear in a repetitive fraction and often carry deleterious
mutations (Teruel et al. 2010). Transcriptional activity of
gene fragments located on Bs has been shown in rye together
with an alteration of the transcription of corresponding gene
copies on A chromosomes (Carchilan et al. 2009).
151
10.2.3 DNA Composition of Rye B
Chromosomes
One of the best studied models in B chromosome research is
the Bs of rye (Secale cereale). Under the microscope the B
of rye is easily distinguishable from the As by eye even
without a specific marker. In contrast to the metacentric
As, the B is acrocentric. Based on Giemsa-banding, the B
appears to have both heterochromatic and euchromatin parts,
while the B-terminal region is Giemsa-staining positive
(Jones and Puertas 1993). The total size of a rye B has
been estimated to be c. 400 Mb as 1C content (Jones et al.
2008b). At interphase, Bs show a predisposition for coori-
entation and a tendency for association, supporting even
numbers of Bs. This association was shown in a background
of hexaploid wheat (Morais-Cecilio et al. 1996) and in rye
(Morais-Cecilio et al. 1997). It has also been suggested that
the Bs of rye influence the ribosomal DNA (rDNA) organi-
zation at interphase. Studies showed that the presence of Bs
increased the condensation state of these regions on the As
suggesting the Bs were influencing the transcription of
rDNA on the A chromosomes (Delgado et al. 1995). In
addition, the presence of B chromosomes was shown to
result in the condensation of an A-located satellite repeat.
These condensation events were not directly proportional to
the number of Bs, suggesting that this effect is caused by the
Bs themselves rather than by the increase of total nuclear
DNA content (Delgado et al. 2004).
At the DNA level, apart from the terminal region of the
long B chromosome arm, a high level of overall similarity
exists between As and Bs of rye (Timmis et al. 1975;
Tsujimoto and Niwa 1992; Wilkes et al. 1995; Houben
et al. 1996). This hints at the intraspecific origin of this
type of chromosome. Also rye Bs appear to be monophyletic
and very stable, as they are very similar even in other rye
species like Secale segetale, which is very closely related to
Secale ancestrale (Niwa and Sakamoto 1995). This is rather
unexpected since Bs are expected to have an elevated muta-
tion rate compared with the A genome.
Using genomic DNA from a plant with no B chromosomes
(0B) as a probe for genomic in situ hybridization (GISH)
resulted in signal labelling along the whole of the Bs except
the heterochromatic terminal part (Tsujimoto and Niwa
1992; Wilkes et al. 1995; Morais-Cecilio et al. 1996). This
region of the B is unstable, prone to rearrangements
and favours duplication over deletion (Langdon et al.
2000). It is enriched in B-specific sequences (Houben et al.
1996) such as D1100 (Sandery et al. 1990; Wilkes
et al. 1995) and E3900 (Blunden et al. 1993). Sequence
comparisons have shown that there are similarities between
152
D1100 and Miniature Inverted-repeat Transposable Element
sequences (MITEs) which appear frequently in temperate
cereals (Langdon et al. 2000). E3900 shows similarities to
Ty3-gypsy retrotransposons and contains a partial reading
frame of a gag gene. A possible ancestor of the gag fragment
that frequently colonizes the centromeres of Poaceae was
cloned and described (Langdon et al. 2000). It therefore
seems likely that members of the D1100 and E3900 families
were formed from parts of the A genome which underwent
frequent amplification and simplification steps (Langdon
et al. 2000).
Several subtelomeric, high copy sequences common to
the As are reported to be missing from the Bs such are pSc74
(Tsujimoto and Niwa 1992; Manzanero and Puertas 2003),
pSc119 (Tsujimoto and Niwa 1992; Wilkes et al. 1995;
Manzanero and Puertas 2003), pSc200 (Hasterok et al.
2002; Kubalakova et al. 2003; Manzanero and Puertas
2003; Zhou et al. 2010) and pSc250 (Kubalakova et al.
2003). However, other authors have claimed a potential
polymorphism on Bs that carry traces of pSc74 and pSc119
in the subtelomeric end of the long arm (Cuadrado and Jouve
1994; Kubalakova et al. 2003). Tsujimoto and Niwa (1994)
proposed that the differential presence of high copy tandem
repeats (satellite DNA) is caused by the different population
genetics of As and Bs. Bs are usually present in only some
individuals in a random mating population, therefore, if a
spontaneous amplification of a satellite sequence occurs in
an individual without the B chromosome, the amplified
sequence will not become distributed over the Bs.
The chromosomal ends of Bs do contain typical
Arabidopsis-type telomere sequences (Manzanero and
Puertas 2003). However, whether ribosomal genes are pres-
ent is still controversial. While Flavell and Rimpau (1975)
reported they were present on rye Bs, more recent studies
have failed to confirm their findings (Cuadrado and Jouve
1994; Kubalakova et al. 2003). The centromeres of both
types of chromosomes are enriched in paWRC.1 sequences,
so-called Bilby repeats (Francki 2001), although a more
extended distribution of Bilby elements has been reported
for Bs (Wilkes et al. 1995). Figure 10.1 summarizes the
DNA composition of rye A and B chromosomes.
Several translocations between rye As and Bs have been
reported although they mostly arise from artificially-induced
events via irradiation (Hasterok et al. 2002) or strains kept
under laboratory conditions for very long times (Schlegel
and Pohler 1994; Wilkes et al. 1995). Indeed, the contra-
dictory findings of the subtelomeric repeat sequences pSc74
and pSc119 (Tsujimoto and Niwa 1992; Cuadrado and Jouve
1994) have been attributed to such translocations (Wilkes
et al. 1995). It has also been found, that these translocations
are not stable in their respective populations (Hasterok et al.
2002). One incident was published where a translocation
spontaneously arose in a strain sampled from wild populations,
A. Houben et al.
which developed a deficient form of the rye B lacking the
B-specific heterochromatic distal part (Ribeiro et al. 2004).
However, apart from that example, only the analysis of a
large number of Bs has enabled sporadic variants to be
identified (Kubalakova et al. 2003). It is notable though
that many of these translocations appear to involve the
A chromosome 1R. Artificially-induced translocations in a
wheat rye B addition line have been useful instruments for
mapping the Bs (Endo et al. 2008). Other structural variants
that arise in experimental strains include iso-chromosomes
which contain only the long arm or only the short arm of the
standard B (Jones and Puertas 1993).
10.2.4 Chromatin Composition
of B Chromosomes
Although chromatin structure is increasingly seen as playing
an essential role in different aspects of chromosome func-
tion, little information is available on the chromatin compo-
sition of Bs, and whether it differs from that of the standard
A chromosomes. Based on classical cytological observations
(e.g., Giemsa-banding) an early survey suggested that the Bs
in about half of the plant species which carry them were
heterochromatic (Jones 1975). Indeed, because no genes
with specific phenotypic effects necessary for normal devel-
opment are known for Bs, the finding that some of them are
totally euchromatic is surprising.
In some species like the mouse Apodemus flavicollis
(Tanic et al. 2005), the snail Helix pomatia (Evans 1960)
and the plants Scilla vvedenskyi (Greilhuber and Speta 1976)
and Allium flavum (Vosa 1973), the Bs are predominantly
euchromatic. At interphase, Bs of some plant species, e.g.,
Anthoxanthum aristatum (Östergren 1947), Puschkinia
libanotica (Barlow and Vosa 1969), Scilla autumnalis
(Ruiz-Rejon et al. 1980), Clematis orientalis and C.
hatherliensis (Shambulingappa 1965), Rosa rugosa (Price
et al. 1981), Tainia laxiflora (Tanaka and Matsuda 1972) or
Picea glauca (Teoh and Rees 1977) display so called hetero-
chromatic chromocenters after Giemsa- or Feulgen-staining.
Recent advances in chromatin characterization, in terms
of epigenetic marks, has shown the involvement of DNA
methylation and post-translational histone modifications in
chromatin assembly and maintenance (Richards and Elgin
2002; Craig 2005; Kouzarides 2007). The N-terminal tails of
the nucleosomal core histones, extending from the nucleo-
some surface, are subjected to post-translational modifi-
cations such as acetylation, methylation, phosphorylation,
ubiquitination, glycosylation, ADP-ribosylation, carbonyla-
tion and sumoylation. Acetylation of histones is mainly
linked with transcriptional activation, DNA recombination
and repair (Grunstein 1997; Struhl 1998; Ikura et al.
2000; Bird et al. 2002). Phosphorylation correlates with
10 Biology and Evolution of B Chromosomes
Fig. 10.1 Comparison of the distribution of high-copy sequences
between the A and B chromosomes of rye: Chromosomes are sche-
matically shown with their Giemsa-banded regions in dark blue. Centro-
meric regions are highlighted for orientation. Dark green bars indicate the
location and density of the corresponding repeats as determined by fluo-
rescent in situ hybridization. Light green indicates contradictory finding
or possible polymorphisms. Name of sequences and corresponding
references: (1) D1100, Sandery et al. (1990); (2) E3900, Blunden et al.
(1993); (3) pSc200, Zhou et al. (2010), Kubalakova et al. (2003),
Manzanero and Puertas (2003); (4) pSc74, Tsujimoto and Niwa (1992),
transcription activation, apoptosis, DNA repair, chromo-
some condensation, sister chromatid cohesion/segregation
and gametogenesis (Kaszas and Cande 2000; Prigent and
Dimitrov 2003; Ahn et al. 2005; Krishnamoorthy et al.
2006; Houben et al. 2007). The most characterized histone
modification to date is methylation. This modification occurs
only on lysine (K) and arginine (R) residues of histone H3
and H4 and is performed by histone methyltransferases
(HMTs) (Martin and Zhang 2005). Each arginine can be
mono- or dimethylated and each lysine can be either
mono-, di-, or trimethylated (Zhang and Reinberg 2001).
153
Cuadrado and Jouve (1994), Manzanero and Puertas (2003); (5) R173,
Wilkes et al. (1995); (6) pAWRC.1, Bilby, Wilkes et al. (1995); (7) pSc34,
Cuadrado and Jouve (1994), Manzanero and Puertas (2003); (8) pSc119.2,
Tsujimoto and Niwa (1992), Cuadrado and Jouve (1994), Kubalakova
et al. (2003) and Manzanero and Puertas (2003); (9) B1334, Carchilan
et al. (2009); (10) B8149, Carchilan et al. (2009); (11) B2465, Carchilan
et al. (2009); (12) Afa, Kubalakova et al. (2003); (13) GAA, Kubalakova
et al. (2003); (14) Arabidopsis-like telomere, Manzanero and Puertas
(2003) and (15) 5S rDNA Kubalakova et al. (2003)
Several studies have shown that modification of the histone
H3 tail by methylation of lysine residues 9 and 27 negatively
regulates transcription by mediating a compact chromatin
structure. In contrast, euchromatin is marked by methylation
of lysine residues 4 and 36 (reviewed by Martin and Zhang
2005). Whereas euchromatin-specific methylation of H3K4
is highly conserved among eukaryotes, heterochromatin
indexing by methylation marks at H3K9, 27 and H4K20 is
more variable (reviewed by Fuchs et al. 2006).
Although chromosome banding results suggest a similar
eu- and heterochromatin composition in the B and A
154
chromosomes of Brachycome dichromosomatica (which car-
ries large and micro Bs), the use of highly specific antibodies
against various lysine residues of histone H3 has shown that
the Bs are characterised by a low level of euchromatic
histone marks. Heteropycnotic, tandem repeat-enriched
micro Bs revealed only traces of these histone modifications.
No differences between As and Bs were found for the hetero-
chromatic marks H3K9me1,2 and H3K27me1,2 indicating
that Bs are not marked by an enriched level of heterochro-
matic histone marks, but rather by a low level of
euchromatin-associated histone modifications (Marschner
et al. 2007a). A comparable distribution of histone marks
was also found for the Bs of Crepis capillaris (Houben et al.
2003) and for the heterochromatic Bs of Puschkinia
libanotica (Kumke et al. 2008).
In rye, the subterminal heterochromatic domain of the B is
characterised by a unique combination of histone methylation
marks (Carchilan et al. 2007). Contrary to the heterochromatic
regions of the A chromosomes, this domain is simultaneously
marked by trimethylated histone H3K4 and trimethylated
H3K27. In addition, this domain shows a dark Giemsa band
at mitosis, but undergoes decondensation during interphase and
reveals transcription of B-specific high copy repeat families.
The distribution patterns along A and B chromosomes
observed for the heterochromatin marks H3K9me1,2 were
mainly uniform. The terminal heterochromatic regions of As
and Bs showed little H3K27me1 but were enriched in di- and
trimethylated H3K27 (Carchilan et al. 2007).
Bs of maize display similar distribution patterns of
signals from H3K9me2, H3K27me2 and H3K27me1 com-
pared with the As. However, the H3K27me2 signal on the B
was less than those on the As. The H3K4me2 distribution
pattern on the B matched the position of cytologically visible
euchromatin. Interestingly, only extremely faint H3K12ac
signals were observed in the Bs (Jin et al. 2008). A reduced
level of histone H3 and H4 acetylation was also shown for
the Bs of the grasshopper Eyprepocnemis plorans (Cabrero
et al. 2007) and of B. dichromosomatica (Houben et al.
1997a), respectively.
Not only histones but also cytosine, which is one of the
four essential base units of DNA can be extensively
methylated. In both animals and plants, cytosine is primarily
methylated at the CG dinucleotide position. However, in
plants methylation is not restricted to the CG sequence:
CHG and the less abundant CHH sequence context are also
possible (Gruenbaum et al. 1981). DNA methylation is
involved in the silencing of transposable elements and genes
(Gehring and Henikoff 2007). The DNA repeats and mobile
elements on Bs are also methylated, as reported for the Bd49
tandem repeat in B. dichromosomatica (Leach et al. 1995) and
E3900 repeat in S. cereale (Langdon et al. 2000). However, a
higher DNA methylation level of B chromosomes has not
been reported for any plant species.
A. Houben et al.
10.3 Effects and Transcripts Associated With
B Chromosomes
Although Bs are not essential, some phenotypic effects have
been reported, and these are usually cumulative, depending
upon the number and not the presence or absence of Bs. In
low numbers, Bs have little if any influence on the pheno-
type, but at high numbers they often have a negative effect
on fitness and fertility of the organism (reviewed in Jones
and Rees 1982; Jones 1995; Bougourd and Jones 1997;
Carlson 2009).
There is evidence that Bs directly or indirectly influence
the behaviour of A chromosomes (as noted above for
rDNA—see Sect. 10.2.3). One of the most striking of such
effects is the potential impact of Bs on diploidization
in allopolyploid hybrids, e.g., Lolium temulentum

L. perenne þ B (Evans and Davies 1985), where Bs were
observed to prevent or suppress the homoeologous pairing of
As. Another example is in wheat
Aegilops hybrids where
Bs contributed by the Aegilops parent seem to be able to
substitute for the Ph1 locus of hexaploid wheat. Further
examples are reviewed by Jenkins and Jones (2004) and
Tanaka and Kawahara (1982).
Indirect evidence for weak transcriptional activity of Bs
comes from the comparative analysis of esterase isozyme
activity in two plants with and without Bs-Scilla autumnalis
(Ruiz-Rejon et al. 1980) and rye (Bang and Choi 1990). In
B-positive plants, additional bands were detected by protein
electrophoresis, however in both cases whether the addi-
tional bands were caused by a B-located gene or whether
Bs influenced the transcription behaviour of an A-located
gene remained unclear. For grasshoppers, Bs have been
demonstrated to alter the expression of A chromosome
genes (Teruel et al. 2007).
Except for the B-located 45S rRNA gene of Crepis
capillaris, in which one of two B-specific members of the
rRNA gene family was weakly transcribed (Leach et al.
2005), there was no direct molecular evidence for tran-
scription of B chromosome genes in plants until the tran-
scriptional activity of B-specific repetitive sequences was
demonstrated. In maize (Lamb et al. 2007b) and rye
(Carchilan et al. 2007) retrotransposon-derived high-copy
elements have been shown to be transcriptionally active. In
addition in rye two repeat families, E3900 and D1100
clustered at the B chromosome long arm, have now been
shown to be transcribed in a tissue-specific manner
(Carchilan et al. 2007). The function of these B-transcripts,
and the mechanism of transcription of B-repeats, are
unknown at present. It has been hypothesized that these
transcripts could have a structural function in the organiza-
tion and regulation of Bs (Carchilan et al. 2007; Lamb
et al. 2007b).
10 Biology and Evolution of B Chromosomes 155

Recently, the general transcription activity of rye Bs was

analysed by comparative cDNA-AFLP analysis (Carchilan
et al. 2009). In addition to weak B chromosome transcrip-
tion, the data showed that Bs were able to down-regulate
A chromosome-localized sequences in a genotype depen-
dent manner. It is likely that Bs may bring about a variety
of epigenetic effects, including the differential regulation of
A-localized transposable elements through mechanisms
such as homology-dependent RNA interference pathways.
Since the rye B most likely originated from the A chromo-
some complement, it seems reasonable to suggest that the
transcription alterations of A-located sequences are caused
by homology-dependent mechanisms, as has been proposed
for the remodelling of gene-activities in newly formed
hybrids and allopolyploids (Comai 2005). Another hypothe-
sis for explaining how the Bs exert control over the rest of
the genome postulates their effects on the spatial organiza-
tion of the genome itself. Recent work suggests that spatial
positioning of genes and chromosomes can influence gene
expression (Misteli 2007). Indeed, Delgado et al. (2004)
observed that in rye interphase nuclei, rDNA located on
A chromosomes had a more compact distribution in cells
with Bs compared with cells without Bs. A more compact
distribution of rDNA sites suggests a lower level of rRNA Fig. 10.2 Mitotic nondisjunction of Brachycome dichromosomatica
(2n ¼ 4 þ Bs) micro B chromosomes. The B-specific tandem Bdm29
gene activity. A similar effect of an almost gene deficient
(Houben et al. 1997b) was used as a probe for fluorescence in situ
chromosome has been demonstrated in Drosophila hybridization. Note the unequal distribution of micro Bs during
melanogaster by Lemos et al. (2008) who showed that the anaphase
Y chromosome of D. melanogaster regulates the activity of
hundreds of genes located on other chromosomes. while the number of Bs in roots is constant, the number of
Bs in aerial organs shows variation (Rutishauser and
R€othlisberger 1966). In grasshoppers variation in Bs has
10.3.1 Mitotic and Meiotic Segregation been observed to occur specifically among follicles of testis
Behaviour of B Chromosomes (Nur 1963, 1969). A detailed list of organisms with unstable
Bs during mitosis is given by Jones and Rees (1982). The
B chromosome inheritance is irregular and non-Mendelian, reason of this numerical variation is shown to be nondisjunc-
and therefore polymorphisms exist with respect to the num- tion of the B chromosome sister chromatids during anaphase
ber of Bs within populations or even within different cell of mitosis leading to an absence of Bs in one daughter cell
lines of an individual carrying Bs. There are several factors and accumulation in the other (Fig. 10.2). If nondisjunction
which affect the rate of transmission of Bs from one cell causes lagging of Bs at anaphase it could also result in a loss
to another and from one generation to another. Drive of B chromosomes in the daughter cells and consequently
mechanisms play a major role in the equilibrium of B fre- exclusion of Bs from those cells.
quency in populations (Jones 1991).
10.3.1.2 Meiotic Behaviour of B Chromosomes
10.3.1.1 Mitotic Behaviour of B Chromosomes B chromosomes fail to pair with any member of the A
In most species which carry Bs, the mitotic transmission of chromosome set during meiosis, although they may pair
Bs during growth and development is disjunctional and and form chiasmata among themselves. However, there are
hence all cells carry the same number of Bs within the several reports indicating that in some species Bs cannot
individual. However, there are some exceptions in which even pair and form chiasmata (Jones and Rees 1982) and
the Bs show instability during mitosis in somatic tissues the exact reason for this is unclear. Nevertheless, Jones and
and therefore they are absent or present in variable numbers Rees (1982) have suggested various hypotheses. One possi-
in specific tissues and/or organs. For example, in the grass bility is that Bs in these species are not homologs of each
Aegilops speltoides Bs exist in aerial organs but not in roots other and therefore cannot pair. However, this implies that
(Mendelson and Zohary 1972), and in Crepis capillaris, all analysed individuals in these studies have been carrying
156
structurally different Bs which is unlikely. The other possi-
bility put forward is that Bs in some species such as Allium
cernuum (Grun 1959) are too short to be able to pair. The
large amount of heterochromatin on Bs has also been
suggested to be another reason for inhibition of chiasmata
formation. However, this is not a general rule as in some
species like Locusta migratoria (Kayano 1971) Bs are het-
erochromatic and yet form mainly bivalents.
Depending on the number, Bs can form uni-, bi- or
multivalents in metaphase I. The formation of bivalents or
multivalents in species with two or more Bs is rarely com-
plete and the formation of univalents among Bs is far higher
than the rate of univalent formation from As (Jones and Rees
1982). The fate of these univalents varies in different species
under diverse conditions. They will usually be lost during
first meiosis or, in the case of the precocious division of
sister chromatids, in second anaphase. Like in Aegilops
speltoides with one B, univalent Bs behave in various ways
in different meiotic cells (Mendelson and Zohary 1972). In
the majority of pollen mother cells univalent Bs will be lost
but in the remaining cells univalent Bs move undivided to
one of the daughter nuclei and separate normally in second
anaphase. Therefore, only around 10% of tetrads carry one B.
In the grasshopper Myrmeleotettix maculatus there is no loss
of Bs as all univalents move undivided to one of the two
telophase nuclei at the first division of meiosis (John and
Hewitt 1965). The study of rye plants with two Bs in
genotypes with low and high levels of B transmission
revealed that the main cause of the difference in the rate of
B transmission was their ability to form B bivalents at
metaphase I. When the Bs formed bivalents they separated
normally in meiosis and were conserved in pollen grains. In
contrast, when they formed univalents they were mainly
eliminated (Jimenez et al. 1997).
There are several scenarios in which univalent Bs can
escape lagging and consequently loss during meiosis. One
way to prevent univalent Bs being lost is by the
incorporation of nondivided univalent Bs into the first telo-
phase nuclei by association of an univalent B with a non-
homologous chromosome. This situation has been observed
in the grasshopper Tetrix ceperoi in which the B and the
X chromosomes form a quasi-bivalent which moves nor-
mally to opposite poles in first anaphase (Henderson 1961).
In the plant Parthenium argentatum Bs form bivalents or
multivalents between themselves via chiasmata during pro-
phase. Then, during metaphase the chiasmata are resolved
and the majority of undivided re-univalented Bs move to
either of the poles and consequently there is no or little loss
of Bs at meiosis (Catcheside 1950). In Plantago serraria the
univalent Bs accumulate near one of the two spindle poles
and consequently remain in one of the telophase nuclei
after anaphase I, and separate normally in anaphase II
(Frost 1959).
A. Houben et al.
10.3.2 Diverse Drive Mechanisms
of B Chromosomes
The variety of mechanisms, including segregation failure by
which Bs gain advantage in transmission are known as accu-
mulation or drive mechanisms. Drive is the key to under-
standing Bs and it occurs in a great variety of ways and for
none of these is the molecular mechanism known (Burt and
Trivers 2006). Since Bs appear to be devoid of essential genes
and have no known adaptive advantage, in many cases, their
maintenance in natural populations is made possible by their
transmission at higher than Mendelian frequencies, and this
enables their successful accumulation in populations. The
importance of drive is that it retains the B in the population
even if its presence has harmful effects on the general viabil-
ity of its bearers (Kimura and Kayano 1961). Depending on
the species, B chromosome drive can be premeiotic, meiotic
and post-meiotic although in all cases the outcome is the same
i.e., higher number of Bs in the next generation.
In animals, as the gametic nuclei are not replicating, the
accumulation mechanism effectively acts either before or
during meiosis. Premeiotic drive mechanisms in animals
occur in the spermatogonial mitosis in the testes (Jones
1991). Based on the observations of instability in the number
of Bs in somatic and germline cells in different species of
grasshoppers, it is inferred that a mitotic nondisjunction
occurs and the cells containing Bs are preferentially included
into germ line cells. Nondisjunction is suggested to happen
early in the embryonic divisions as in Calliptamus palaes-
tinensis (Nur 1963) or later on like in Locusta migratoria
(Kayano 1971). Premeiotic drive has also been described in
the plant Crepis capillaris with the difference that mitotic
nondisjunction occurs in the meristematic cells at the time
of flower initiation and with the preferential inclusion of
Bs in inflorescences during development (Rutishauser and
R€othlisberger 1966; Parker et al. 1989). The preferential
inclusion of Bs in inflorescences coincides with asymmetri-
cal spindles which occur in shoot meristems during the tran-
sition from vegetative to reproductive growth (Rutishauser
and R€othlisberger 1966).
Meiotic drive in the megaspore mother cell has been clearly
shown in Lilium callosum (Kayano 1957), a species with
tetrasporic embryo sac development. Here, univalent Bs in
egg mother cells tend to be unevenly distributed. Rather than
lying on the metaphase plate of metaphase I, univalent Bs were
observed to be located on the micropylar side of the spindle in
80% of the analysed cells resulting in their preferential
incorporation into the resulting egg cell. A similar situation
was observed for the grasshopper Myrmeleotettix maculatus in
which the Bs were preferentially locating at the egg pole rather
than polar bodies pole. Although in the males the Bs were lost
during meiosis due to lagging, overall there was a net meiotic
drive acting on Bs in M. maculatus (Hewitt 1976).
10 Biology and Evolution of B Chromosomes
standard B
deficient B
long iso-B
short iso-B
translocated wheat/rye B
translocated wheat/rye B
directed nondisjunction of B
a b c d
f g
Fig. 10.3 Diagram showing the function of the distal region of the long
chromosome arm and the pericentromeric sites in the process of directed
nondisjunction of the rye B during first pollen mitosis. The standard B has
pericentromeric sticking sites (grey blocks) and a heterochromatic distal
region on the long arm which contains the controlling region for nondis-
junction (red blocks). The translocated A/B chromosome is shown in
green and blue for wheat and rye chromatin, respectively. (a) Sister
chromatids of a standard B (Hasegawa 1934) and (b) of a B long arm
isochromsome (M€untzing 1948). (c) If standard and deficient Bs are both
present, both B-types show directed nondisjunction (Lima de Faria 1962).
Post-meiotic drive is frequent in flowering plants during
male gametophyte maturation. The drive mechanisms of
maize and rye Bs are well-studied examples that result in
B chromosome accumulation. In rye, directed nondisjunc-
tion happens during first pollen grain mitosis resulting in the
accumulation of Bs in the generative nucleus and therefore
ensuring their transmission at a higher than expected rate to
the next generation (Hasegawa 1934). Notably, the B cen-
tromere appears to divide normally, but on either side are
“sticking sites” which prevent normal anaphase separation
of the chromatids and results in nondisjunction at an average
frequency of about 86% (Matthews and Jones 1983). In the
second pollen grain mitosis the generative nucleus divides to
produce two sperm nuclei, each with an unreduced number
of Bs. A similar nondisjunction process may occur in the
female line as well (Hakansson 1948). Interestingly, nondis-
junction works equally well when the rye B is introduced as
157
the controlling region for nondisjunction,
location of B-specific E3900 and D1100
repeats
rye chromatin
wheat chromatin
pericentromeric chromatid adhesion sites
vegetative pole generative pole
disjunction of B
e
rye
h
wheat/rye B
addition lines
(d) A chromosome which is deficient for the distal part of the long B arm
(Lima de Faria 1962) or (e) a short arm isochromosome shows normal
disjunction (M€ untzing 1948). (f) Directed nondisjunction is independent
from the background genotype as a standard rye B shows directed nondis-
junction if added to wheat (Endo et al. 2008). (g) In the case of a reciprocal
A/B chromosome translocation, the chromosome possessing the B cen-
tromere shows directed nondisjunction. Hence, nondisjunction functions
in trans (Endo et al. 2008). (h) Translocated A/B chromosome without the
distal heterochromatic block of the B shows normal disjunction (Endo
et al. 2008)
an addition chromosome into hexaploid wheat (M€ untzing
1970; Niwa et al. 1997; Endo et al. 2008) or Secale vavilovii
(Puertas et al. 1985). Therefore, the segregation behaviour of
the B is mainly autonomous and independent of the back-
ground genotype. The B itself controls the process of non-
disjunction and B-transmission frequency (Romera et al.
1991). The accumulation mechanism of the rye B requires
a factor located at the end of its long arm as Bs which lack
this terminal region (where two B-specific repeat families
E3900 and D1100 reside) undergo normal disjunction
(Fig. 10.3a, d) (M€untzing 1948; Endo et al. 2008). This
factor may act in trans because if a standard B (Fig. 10.3c)
(Lima de Faria 1962) or the terminal region of the long arm
of the B (Endo et al. 2008) is also present in the same cell
containing a B lacking the terminal region then the standard
B mediates nondisjunction of both itself and the deficient B
(Fig. 10.3c). Interestingly, it has been shown that D1100 and
158
E3900 are transcriptionally active with higher expression
levels in the anthers (Carchilan et al. 2007).
So far no gene, or other DNA sequence on any B has been
characterized that plays a role in B chromosome accumula-
tion, and it is therefore tempting to speculate that non-coding
RNA is involved in the process of B chromosome nondis-
junction. In fission yeast, for example, the repeats flanking
the centromere are essential for sister chromatid cohesion
and are maintained in a proper heterochromatic state by the
RNAi machinery (Volpe et al. 2003). Similarly, pericen-
tromeric heterochromatin is required for proper chromo-
some cohesion and disjunction in flies and other organisms
(Pidoux and Allshire 2005; Vos et al. 2006). It is also noted
that the forced accumulation of human centromeric non-
coding satellite transcripts leads to defects in the separation
of sister chromatids (Bouzinba-Segard et al. 2006) and in
plants, RNA molecules have been shown to play a role in
establishing centromeric heterochromatin domains (Topp
et al. 2004; May et al. 2005). In this context, the transcrip-
tional activity of the D1100 and E3900 repeats (noted above)
located in the B chromosome nondisjunction-controlling
region of rye is striking (Carchilan et al. 2007). However,
no similarity between D1100/E3900 repeats and B-
centromere located sequences has been found based on
fluorescent in situ hybridization (FISH) experiments
(Houben et al. unpublished). Nevertheless, it is noted that
sequence similarity between non-coding RNA and a target
region does not seem to be a functional requirement. For
example, dosage compensation in both flies and mammals
requires non-coding RNAs which spread in cis to coat the X
chromosome. The regions of the Xist RNA that are required
for localization on the Xi have no obvious sequence homol-
ogy (Wutz et al. 2002). This also holds true in the case of
Drosophila roX RNAs, which contain little sequence homol-
ogy to one another, except for a stretch of 30 nt (Meller and
Rattner 2002).
In maize the nondisjunction process differs from that in
rye. At least three properties allow the maize B to increase in
numbers: nondisjunction at the second pollen grain mitosis,
preferential fertilisation of the egg by sperm containing B
chromosomes (Roman 1948; Carlson 1969; Rusche et al.
1997), and suppression of meiotic loss when the Bs are
unpaired (Carlson and Roseman 1992). The lack of meiotic
loss of B univalents is a special feature of maize Bs. In rye,
for example, the B univalents are lost in about 80% of
1B
0B crosses (Jimenez et al. 1997). As in rye, the B-
accumulation mechanism in maize requires a factor located
on the end of the long arm of the B that may act in trans
(Roman 1947; Carlson 1978; Lamb et al. 2006). Further-
more, nondisjunction of Bs takes place in the endosperm and
in the tapetum. In binucleated tapetal cells, Bs mediate A
chromosome instability (Chiavarino et al. 2000; Gonzalez-
Sanchez et al. 2004). Sporophytic nondisjunction of Bs
A. Houben et al.
occurs if Bs are present at high copy number which could
imply that nondisjunction is either repressed at low numbers
or so infrequent that it was not observed (Masonbrink and
Birchler 2010). One A-located factor seems to codetermine
maize B accumulation by preferential fertilization while
another factor(s) determines the meiotic loss of Bs
(Gonzalez-Sanchez et al. 2003). Sperm nuclei containing
deletion derivatives of B-9 (translocation lines involving
the B and chromosome 9), which lack the centric hetero-
chromatin and possibly some adjacent euchromatin of the B
chromosome, no longer have the capacity for preferential
fertilization (Carlson 2007).
Generally there are two models for explaining the mech-
anism of directed B chromosome nondisjunction in rye
(Jones 1991). In the active model, the chromatids of Bs are
delayed in their separation and an active movement prefer-
entially takes the Bs to the generative nucleus. In the passive
model, the movement of Bs towards the generative nucleus
is simply caused by the fact that the equatorial plate is closer
to the generative pole and lagging Bs are included in the
generative nuclei as the nuclear membrane is formed. In
future, the potential interrelationship between unequal cell
division and directed nondisjunction should be experimen-
tally addressed. In addition, studies on artificial chromo-
somes might help to understand the factors affecting B
chromosome segregation. As different artificial mammalian
chromosomes missegregate over a five-fold range, the data
suggest that variable centromeric DNA content, kinetochore
composition and/or epigenetic assembly as well as topo-
logical positioning in the nucleus can influence the mitotic
segregation behaviour of chromosomes (Rudd et al. 2003;
Moralli et al. 2009). The presence of pericentromeric hetero-
chromatin, checkpoints, sister chromatid cohesion or other
yet unidentified components may be necessary for B chromo-
some drive, and one or more of these aspects may be
different in the Bs.
10.4 Evolution of B Chromosomes
Several scenarios have been proposed for the origin of Bs.
Most probably they have arisen in different ways in different
organisms, see reviews by Camacho et al. (2000), and Jones
and Houben (2003). The most widely accepted view is that
they are derived from the A chromosome complement.
Some evidence also suggests that Bs can be spontaneously
generated in response to the new genomic conditions
after interspecific hybridization. The involvement of sex
chromosomes has also been argued for their origin in some
animals, see Camacho et al. (2000) for examples. Despite the
large number of species with Bs, their de novo formation is
probably a rare event because the occurrence of similar B
variants in related species suggests a close relationship
10 Biology and Evolution of B Chromosomes
between the different variants, e.g., in Secale (Jones and
Puertas 1993) and Brachycome (Houben et al. 1999).
The molecular processes that gave rise to Bs during
evolution remain unclear, but the characterization of
sequences residing on them has helped to shed some light
on their origin and evolution. In maize and Brachycome
dichromosomatica for example, the Bs contain sequences
that originate from several different A chromosomes, so the
Bs could represent an amalgamation of these diverse A-
derived sequences (Alfenito and Birchler 1993; Houben
et al. 2001b; Cheng and Lin 2003; Peng et al. 2005).
The actual process of sequence transfer from As to Bs is
not clear, but recent results indicate that transposition of
mobile elements may have played an important role (Cheng
and Lin 2004; Lamb et al. 2007b; Carchilan et al. 2009). For
example, an analysis of large DNA insert clones has shown
that maize Bs are composed of B-specific sequences that are
intermingled with those in common with the As. The 22 kb-
long B-specific StarkB element, for example, has been subject
to frequent insertions by LTR-type retroelements (Lamb et al.
2007b), in a fashion similar to the nested insertions seen in
some intergenic A chromosome regions (SanMiguel et al.
1996). It therefore seems likely that the StarkB tandem array
formation started with the transposition of a GrandeB mobile
element into an original non-GrandeB sequence, which was
then amplified, possibly from sister-strand exchange. Subse-
quently invasion(s) together with amplification resulted in the
loss of the original tandem structure of StarkB (Lo et al.
2009). Using the divergence of retroelements interrupting B-
specific sequences, Lamb et al. (2007b) have estimated the
minimum age of the maize B to be at least 2 million years.
The recently established oat-maize B chromosome addition
lines (Kynast et al. 2007) are an ideal material to further
characterize the sequence composition of the maize B chro-
mosome because of the low level of sequence similarity
between oat and maize.
B chromosomes provide an ideal target for transposition
of mobile elements (McAllister 1995), and the insertion of
such elements may therefore be responsible for generating
the structural variability observed in Bs (Camacho et al.
2000). Indeed, a B-specific accumulation of Ty3/gypsy
retrotransposons has been reported for the fish Alburnus
alburnus (Ziegler et al. 2003). In the same context, it is
noted that Bs also contain various types of coding and
noncoding repeats which are similar to those found in circu-
lar extrachromosomal DNA of various organisms (Cohen
and Segal 2009). For example, extrachromosomal DNA
with similarity to tandem repeat sequences shared by A
and B chromosomes of B. dichromosomatica has recently
been identified (Cohen et al. 2008), and integration of extra-
chromosomal DNA into Bs may be favoured because tran-
scriptionally less active Bs are subject to reduced negative
selection. However, whether an evolutionary link exists
159
between extrachromosomal DNA and the evolution of Bs
remains to be determined.
The involvement of rRNA coding repeats in the evolution
of B chromosomes does not appear to be accidental, because
rDNA loci have been detected on Bs of many species of both
plants (e.g., Crepis capillaris (Maluszynska and Schweizer
1989)) and animals (e.g., Rattus rattus (Stitou et al. 2000),
Trichogramma kaykai (van Vugt et al. 2005); for review see
Green (1990) and Jones (1995)). In the herb Plantago
lagopus the origin of a B chromosome seems to be associated
with the massive amplification of 5S rDNA sequences after
chromosome fragmentation of an aneuploid A chromosome
(Dhar et al. 2002). Alternatively, but less likely, the location
of rDNA sites on B chromosomes could be a consequence of
the reported mobile nature of rDNA (Schubert and Wobus
1985) with B chromosomes as the preferred ‘landing sites’
due to their neutral character. There is also increasing evi-
dence that ribosomal sequences can change position within
the genome without corresponding changes in the
surrounding sequences (Dubcovsky and Dvorak 1995;
Shishido et al. 2000; Datson and Murray 2006). However, it
is noted that not all Bs carry rDNA.
Except for the 45S rRNA gene on the B chromosome of
C. capillaris, there has been no direct molecular evidence
demonstrating transcription of B chromosome rDNA in any
plant species (Leach et al. 2005). The reason(s) why most
rDNA on B chromosomes is not or only weakly transcribed
is not clear yet although differences in histone H3 methyla-
tion between A and B chromosomes may be responsible
(Marschner et al. 2007a). Another possibility is that suppres-
sion may occur because of nucleolar dominance, so that the
rRNA genes on the As are active at the expense of those on
the Bs (Donald et al. 1997).
Available sequence information for B-located rRNA genes
has been used to study the likely origin of Bs by determining
the relatedness of the internal transcribed spacers (ITS)
between the different chromosome types. For example, analy-
sis of ITS sequences from the A and B chromosomes of
C. capillaris (Leach et al. 2005) and B. dichromosomatica
(Donald et al. 1997; Marschner et al. 2007b), and comparisons
with sequences from related species, indicate that the B chro-
mosome rRNA genes are most likely derived from those of
the A chromosomes in the host species.
Some findings imply that B chromosomes arise spontane-
ously in response to genomic stress following interspecific
hybridization, e.g., in Coix aquatica and C. gigantea (Sapre
and Deshpande 1987). After fertilization, the two different
parental genomes are combined within a single nucleus,
which in most cases is embedded within the maternal cyto-
plasm. Such a novel genomic constitution may result in
conflicts, and as a consequence genomic and epigenetic
reorganization of the genomes can occur (Riddle and
Birchler 2003; see also Jones and Langdon 2013, this
160
volume). An incomplete loss of one parental genome during
hybrid embryogenesis might play a role in the hybrid origin
of Bs. Indeed, there is evidence showing that during the
uniparental chromosome elimination process, the centro-
meres of parental chromosomes undergoing elimination are
the last to be lost (Gernand et al. 2005). If such a centric
fragment is retained, rather than eliminated, a subsequent
spontaneous doubling could provide the ideal starting point
for the de novo formation of a supernumerary chromosome.
Indeed, a centric fragment was generated during the intro-
gression of a chromosome region from the wasp Nasonia
giraulti into N. vitripennis. This neo-B showed a lower than
normal Mendelian segregation ratio in meiosis, and some
mitotic instability; but the transmission rate and mitotic
stability then increased over successive generations (Perfectti
and Werren 2001).
A novel mechanism for chromosome evolution based on
recombination of nonhomologous chromosomes during the
DNA double-strand repair process at S-phase has been
postulated for the formation of the “zebra” chromosome,
which is composed of Elymus trachycaulus/Triticum
aestivum structurally rearranged chromosome fragments
(Zhang et al. 2008). Although this restructured chromosome
does not represent a B chromosome, it is possible to envisage
that a similar mechanism could also result in the formation
of a neo-B chromosome.
On the basis of new insights into the mechanisms of
chromosome evolution (Hall et al. 2006; Lysák et al. 2006;
Schubert 2007; see also Lysák and Schubert 2013, this
volume) we are tempted to ask, as did Patton (1977),
whether the “by-product” of a Robertsonian translocation
between two nonhomologous acrocentric chromosomes
with breakpoints close to centromeres could evolve into a
B-like chromosome. With the recent development of the
comparative chromosome painting technique to reconstruct
karyotype evolution, it is becoming clear that chrom-
osome number reductions are often accompanied by
pericentromeric inversions and translocations between acro-
centric chromosomes (Mandakova and Lysák 2008).
Although the minichromosomes formed from Robertsonian
translocation events are mainly composed of centromeric
sequences, they are frequently lost because of the lack of
essential genes and their failure to pair and to segregate
properly during meiosis. Centromeric regions are also highly
dynamic in sequence composition and display a low recom-
bination frequency (Gaut et al. 2007). Recent findings by
Hall et al. (2006) point to (peri)centromeres as genomic
regions that may experience selective pressures distinct
from those acting on euchromatin. They can tolerate rapid
changes in structure and sequence content, such as large
insertions of sequences that are typical of B chromosomes,
e.g., mobile elements, rDNA arrays, and satellite arrays.
When a nonessential centromeric fragment survives, rapid
A. Houben et al.
sequence alteration may prevent meiotic pairing with the As,
and the gain of a drive (by an unknown mechanism) may put
it on the evolutionary pathway to a proto-B.
In addition, tertiary trisomics, which appear in the
progenies of translocation heterozygotes, have been
hypothesized, under certain circumstances (e.g., suppressed
crossing-over, rapid loss of genetic activity to overcome
genetic imbalance, and positive selection for plants with an
extra chromosome), to be suited for B chromosome forma-
tion, e.g., in the garden pea (Berdnikov et al. 2003).
The most widely used approaches to study the evolution
and DNA composition of B chromosomes are based on the
isolation and characterization of only a single or a small group
of mainly repetitive sequences. These approaches have been
valuable in tracing the fate of various repeats in a wide range of
species. However, they do not allow for the global compara-
tive analysis of sequence profiles required for elucidating
evolutionary trends at the whole genome and B chromosome
level. To overcome this obstacle in the future, new low-cost
sequencing technologies such as 454 pyrosequencing
(Margulies et al. 2005) should be used for B chromosome
sequence analysis. Recently, 454-sequencing was used to
sequence flow sorted plant chromosomes. As few as 10,000
copies of barley chromosome 1H were flow-sorted and used as
a template to assess gene content and genomic composition of
this chromosome (Mayer et al. 2009). As most Bs are smaller
than As, they should be easy to sort by flow cytometry
(Kubalakova et al. 2003) and thus seem readily suited for
such approaches. For instance, one rye B consists of around
560 Mb, so a single 454-run could identify ~50% of the
sequence information of flow-sorted Bs. In the future, the
application of next generation sequencing technologies to
flow-sorted Bs is likely to generate the amount of sequence
data needed for extensive comparative sequence analyses.
This will then significantly improve our knowledge of the
origin of Bs and hence of the evolution of genomes.
Note added in proof Recently, Martis et al. (2012)
reported the results of using 454 pyrosequencing to analyse
flow sorted A and B chromosomes from rye. They observed
that the rye B chromosome is a mosaic of sequences derived
from the A chromosomes as well as the organelles.
Acknowledgments The authors have been supported by the DFG (HO
1779/14-1, HO 1779/10-1).
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 Chromosomes and Sex Differentiation
11
Bohuslav Janoušek, Roman Hobza, and Boris Vyskot

Contents 11.1 Evolution of Plant Sexuality

11.1 Evolution of Plant Sexuality . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Plant species can be classified into two major groups: those
11.2 Sex Chromosomes and Sex Determination Systems
in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 that permit self-pollination (autogamy) and those that inhibit
11.2.1 Sex Determination and Sex Chromosomes in Bryophytes 168 self-pollination. In mostly self-pollinating species, harmful
11.2.2 Sex Determination in Ferns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 recessive mutations with a large effect are efficiently elimi-
11.2.3 Gymnosperms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 nated by selection, while slightly deleterious mutations
11.2.4 Angiosperm Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
accumulate as a consequence of the reduced effective popu-
11.3 Sexual Dimorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 lation size and effective recombination rates (Wright et al.
11.4 The Current Status of Research and Prospects 2008). In contrast, plants that prevent autogamy are able
for the Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 to mask and retain in their genomes harmful recessive
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 mutations with large effects in spite of more efficient selec-
tion against slightly deleterious mutations in this group. In
cosexual plants, various mechanisms, such as dichogamy,
heterostyly or self-incompatibility, prevent self-pollination.
Another mechanism is the evolution of unisexual flowers.
Populations can be distinguished according to the locali-
zation of unisexual flowers: monoecious (male and female
on the same plant), gynomonoecious (hermaphrodite and
female flowers on the same plant), andromonoecious (male
and hermaphrodite flowers on the same plant), dioecious
(male and female flowers on different plants), gynodioecious
(female and cosexual individuals), androdioecious (male
and cosexual individuals), or trioecious (male, female,
and cosexual individuals), as reviewed by Dellaporta and
Calderon-Urrea (1993). Gymnosperms are mostly monoe-
cious, but also comprise a relatively high percentage
of dioecious species. In the gymnosperms, approximately
36 %, namely all c. 250 species of cycads, Ginkgo biloba,
and c. 50 Gnetales are dioecious (Ming et al. 2011). In
contrast, dioecy has been reported in only about 6 % of
angiosperm species (Renner and Ricklefs 1995). Interest-
ingly, dioecy is more widespread in tropical species, and
an exceptionally high percentage of the dominant woody
species of tropical forests are dioecious (Matallana et al.
B. Vyskot (*) 2005). New cases of dioecy continue to be found because
Laboratory of Plant Developmental Genetics, Institute of Biophysics,
Academy of Sciences of the Czech Republic, Kralovopolska 135,
of the phenomenon of cryptic dioecy (Mayer and
Brno 61265, Czech Republic Charlesworth 1991).
e-mail: vyskot@ibp.cz

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 167

DOI 10.1007/978-3-7091-1160-4_11, # Springer-Verlag Wien 2013
168
The taxonomic spread of dioecy indicates that it probably
originated many times independently. There are several
hypotheses that describe the possible evolutionary history
of dioecy. The most popular one is that two basic genetic
changes had to occur on the way from cosexuality to dioecy
(Charlesworth and Charlesworth 1978). One such change
caused male sterility and the other was responsible for
female sterility. Gynodioecy was likely the intermediate
stage towards complete dioecy, since it occurs much more
frequently than androdioecy. According to theoretical stud-
ies, the evolution of dioecy through androdioecy is also
hindered by males in androdioecious populations having to
produce at least twice the pollen of hermaphrodites to be
maintained in the population (Charlesworth and Charlesworth
1978). Other possible routes to dioecy include direct evolu-
tion of dioecy from monoecy, e.g., species of the genus
Siparuna (Renner and Won 2001), or evolution of dioecy
as a reaction to the loss of autoincompatibility in polyploids
(Miller and Venable 2000). The possibility of the evolution
of dioecy through heterodichogamy is also theoretically
supported (Pannell and Verdú 2006), but there is still no
clear example of this route (Renner et al. 2007).
In most dioecious species the male is the heterogametic sex
(XY), while females are homogametic (XX). However, there
are some exceptions, in which female individuals
are heterogametic (ZW), e.g., wild Fragaria (Spigler et al.
2008) and Populus trichocarpa (Tuskan et al. 2006). In some
species, sex is determined by a simple Mendelian genetic
system based on the segregation of a few loci such as in
Fragaria virginiana (Spigler et al. 2008), Ecballium elaterium
(reviewed in Mather 1949; Gómez-Campo and Casas-Builla
1965), Mercurialis annua (Hamdi et al. 1987). In others, such
as Carica papaya, a short X-Y non-recombining region has
recently formed (Yu et al. 2007). These systems are consid-
ered to be evolutionary very young. Other dioecious species
are evolutionary older, and some of them have evolved het-
eromorphic sex chromosomes. Heteromorphic sex chromo-
somes occur in Silene (reviewed by Nicolas et al. 2005) and
sorrel (Rumex spp.; reviewed by Ainsworth et al. 1999), some
hop species (Humulus spp.; reviewed in Shephard et al. 2000),
hemp (Cannabis sativa; reviewed by Menzel (1964) and
Sakamoto et al. (2000)), and some Cucurbitaceae species
(Kumar and Viseveshwaraiah 1952).
11.2 Sex Chromosomes and Sex
Determination Systems in Plants
11.2.1 Sex Determination and Sex
Chromosomes in Bryophytes
11.2.1.1 Monoecious Bryophytes:
Intragametophytic Selfing
Self-fertilization has a different adaptive significance and
effects in species with combined sexes than in those with
separate sexes. In haploid-dominant species such as liverworts,
B. Janoušek et al.
mosses, and hornworts, self-fertilization in the broad sense
(intergametophytic selfing—mating of gametophytes derived
from the same spore) is possible in species with combined or
separate sexes (monoecious and dioecious, respectively).
Monoecious species also possess an additional mode of selfing
(intragametophytic selfing) that leads to complete homozygos-
ity in one step. Eppley et al. (2007) found that while there were
deficiencies of heterozygotes compared to the null expectation
in both monoecious and dioecious mosses, monoecious spe-
cies had significantly higher levels of heterozygote deficiency
than dioecious species. Estimates of selfing rates have
suggested that selfing occurs frequently in monoecious
populations, but only rarely in dioecious populations. How-
ever, significant indications of mixed mating or biparental
inbreeding were found in many populations of two dioecious
species (Polytrichadelphus magellanicus and Breutelia
pendula) (Eppley et al. 2007).
11.2.1.2 Dioecious Mosses and Liverworts
(Marchantia polymorpha Main Model)
The sex chromosomes in liverworts and mosses play a direct
role in development of gametophyte sex organs. Marchantia
polymorpha is a model species that has allowed researchers
to better understand sex determination in bryophytes and to
reveal similarities in the evolution of sex chromosomes
between evolutionary distant species. The dominant phase
of the Marchantia polymorpha life cycle is the haploid
gametophyte, which is either male or female. The male
possesses a Y chromosome, while the female possesses
an X. Asexual propagules (gemmae) are produced by the
thallus, so sex-specific cultures can be easily maintained.
This experimental system allows researchers to monitor the
functions of sex-linked genes without interference from the
other sex chromosome.
The X and Y chromosomes are heteromorphic; they differ
in size, which has made them good candidates for FISH
mapping, permitting direct identification of X- and Y-
located sequences. Okada et al. (2000) constructed genomic
libraries of male and female plants, and isolated seventy
putative male-specific PAC clones based on different
intensities of their hybridization with male and female
DNAs. Y-specificity of one clone (pMM4G7) was confirmed
by Southern blots, PCR analysis and FISH (Okada et al.
2000). Another six male-specific clones were isolated
using representational difference analysis (Fujisawa et al.
2001). A detailed analysis of some of the Y-specific clones
(pMM4G7 and pMM23-130 F12) revealed many repetitive
motifs organised in long stretches. Within these specific
repeats, a novel gene family (ORF162) was described,
which is specifically expressed in male sexual organs and
contains a RING motif (Okada et al. 2001). RING finger
proteins are known to participate in transcriptional repres-
sion and in the ubiquitin-mediated protein turnover pro-
cesses (Borden 2000). Another five genes amplified on the
Y chromosome were described by Ishizaki et al. (2002).
11 Chromosomes and Sex Differentiation
One of the five putative genes shows similarity to a male
gamete-specific protein of lily (Lilium longiflorum) and is
expressed predominantly in male sex organs, suggesting that
this gene has a male reproductive function.
In light of this evidence, Ishizaki et al. (2002) have
suggested that the Y chromosome evolved by co-amplification
of protein-coding genes with unique repeat sequences.
Y chromosomes are different from other chromosomes
because they do not undergo recombination. Yamato et al.
(2007) reported the gene organization of the Y chromosome
in M. polymorpha. On the 10 Mb Y chromosome, 64 genes
were identified, 14 of which were detected only in the
male genome. These genes are expressed in reproductive
organs but not in the vegetative thalli, suggesting their
participation in male reproductive functions. Another 40
genes on the Y chromosome are expressed in the thalli and
the male sexual organs. At least six of these genes have
diverged from their X-linked counterparts that are expressed
in the thalli and sexual organs in female plants, suggesting that
these X- and Y-linked genes have essential cellular functions.
These findings indicate that the Y and X chromosomes share
the same ancestral autosome and support the prediction that
in a haploid organism essential genes on the sex chromosomes
are more likely to persist than in a diploid organism (Yamato
et al. 2007; see also Rensing et al. 2013, this volume).
11.2.2 Sex Determination in Ferns
11.2.2.1 Homosporous Ferns (Main Model
Ceratopteris richardii)
Environmental sex determination (ESD) is the rule in homo-
sporous ferns (Korpelainen 1998). Sporophytes (the diploid
generation) produce free-living gametophytes (the haploid
generation) that are potentially bisexual, i.e., they can bear
female (archegonia) and male (antheridia) reproductive
organs (gametangia) on the same individual. Several envi-
ronmental factors influence sexual expression. One class of
factors, maleness-inducing pheromones, or ‘antheridiogens’
(Schneller et al. 1990), has received much attention.
Antheridiogens are gibberellin-like compounds secreted by
large, female or bisexual gametophytes that reduce growth
and induce maleness in nearby asexual gametophytes.
Other environmental factors that affect sex in ferns, such
as nutrients, have received very little attention in most
reviews of sex expression in fern gametophytes (Cousens
et al. 1988; Korpelainen 1998; see however Raghavan
1989). Three well known patterns described in gameto-
phyte biology illustrate the plasticity of sex expression
in homosporous ferns. Firstly, ontogenetic changes in
gametophyte sex undergo sequential hermaphroditism,
i.e., the unidirectional change in sex during ontogeny.
Klekowski (1969) distinguished four types of sequential
169
hermaphroditism: male to bisexual, male to female to
bisexual, female to bisexual, and step-wise change from
male through bisexual to female. Secondly, the coexistence
of gametophytes of different sexes within a population
has been repeatedly reported (Klekowski 1969; Hamilton
and Lloyd 1991). Thirdly, harsh growing conditions, such
as poor substrate or high density, are known to lead to
small and male gametophytes (Miller 1968; Rubin and
Paolillo 1983; Rubin et al. 1985; Korpelainen 1995;
Huang et al. 2004).
Ceratopteris richardii has been the main fern model for
sex determination studies because of its rapid life cycle and
easy cultivation (Hickok et al. 1987). Haploid gametophytes
of the fern Ceratopteris are either male or hermaphroditic.
The determinant of sex type is the pheromone antheri-
diogen, which is secreted by the hermaphrodite and directs
male development of young, sexually undetermined game-
tophytes. Three phenotypic classes of mutations that affect
sex-determination have been isolated and include the her-
maphroditic (her), the transformer (tra) and feminization
(fem) mutations. Eberle and Banks (1996) performed link-
age analysis and tests of epistasis among the different
mutants to assess the possible interactions among these
putative genes. Their results indicate that sex determination
in Ceratopteris involves at least seven interacting genes,
which may interact with antheridiogen, the primary sex-
determining signal.
Two models describing how antheridiogen may influence
the activity states of these genes and the sex of the gameto-
phyte have been suggested (Talmor-Neiman et al. 2006). To
understand how antheridiogen represses the development of
female traits at the genetic level, 16 new mutations that
feminize the gametophyte in the presence of antheridiogen
were characterized (Strain et al. 2001). Seven are tightly
linked to the FEM1 locus, which was previously described
by Eberle and Banks (1996). Nine other mutations concern
another locus NOTCHLESS1 (NOT1), with mutant plants
lacking a meristem notch. Some not1 mutations also affect
sporophyte development when homozygous, indicating
that the not1 mutations are recessive and that NOT1 is
required for normal sporophyte development. The epistatic
interactions among FEM1, NOT1, and other sex-determining
genes have been revealed (Strain et al. 2001). According to
the current model of sex determination in Ceratopteris, the
presence of antheridiogen leads to the activation of the
FEM1 gene, which not only promotes the differentiation of
male traits, but also represses female development by
activating the NOT1 gene. NOT1 represses the TRA genes
necessary for the development of female traits in the game-
tophyte (Strain et al. 2001).
Kamachi et al. (2007) examined the effect of photomor-
phogenically active light on antheridiogen-induced male
development of gametophytes of Ceratopteris richardii.
170
These workers also revealed the latent antheridiogen-signal
transduction pathway. Although blue light did not affect
sensitivity to antheridiogen in wild-type gametophytes, it
was found that the gametophytes of the her1 mutant, which
are insensitive to antheridiogen, developed into males when
grown under blue light in the presence of antheridiogen. The
latent antheridiogen-signal transduction pathway is therefore
probably activated by blue light. Red light, on the other
hand, suppressed male development, and the action of red
light seems to dominate that of blue light. The results of
experiments with a photomorphogenic mutant also suggest
that phytochrome may be involved in the action of red light
(Kamachi et al. 2007). Interestingly, while certain KNOX
genes function similarly in the development of both seed
plant and fern sporophyte meristems despite their differences
in structure, KNOX gene expression is not required for the
development of the fern gametophyte. It is therefore sup-
posed that the sporophyte and gametophyte meristems of
ferns are not regulated by the same developmental
mechanisms at the molecular level (Sano et al. 2005).
A systemic gene-silencing method suitable for high through-
put, reverse genetic analyses of gene function in fern
gametophytes has already been developed (Rutherford
et al. 2004). This also opens new possibilities for research
into sex determination in ferns.
11.2.2.2 The Heterosporous Ferns and Lycophytes
In contrast to homosporous ferns, heterosporous ferns (e.g.,
Marsilea, Azolla) and some lycophytes (e.g., Selaginella and
Isoetes) form two different types of spores: microspores,
which produce male gametophytes, and megaspores which
produce female gametophytes. These spores are formed in
special structures (microsporangia and megasporangia). In
contrast to dioecious bryophytes where the gametophyte
type is determined by its genome, the gametophyte type in
heterosporous ferns and lycopods is controlled by the type of
spore from which it emerged (reviewed in Tanurdzic and
Banks 2004). A model for the evolution of heterospory from
homospory has been suggested by Haig and Westoby
(1988). This model has three phases: (1) a gradual increase
in spore size in a homosporous population, (2) the sudden
introduction of smaller microspores from sporophytes
reproducing predominantly as males, (3) the subsequent
divergence in size and specialization of the two spore
types. The model proposes that haploid dioecy evolved
from pre-existing mechanisms of sex determination, and
that endosporic development of megagametophytes arose
as a consequence of an increased dependence on spore
food reserves for reproduction (Haig and Westoby 1988).
Among the lycophytes, Selaginella has perhaps the
greatest potential as a useful comparative system for the
study of sex determination and the mechanisms leading to
the origin of heterospory in plants because many species in
B. Janoušek et al.
this genus have small genome sizes permitting the efficient
use of molecular genetic techniques (reviewed by Tanurdzic
and Banks 2004).
11.2.3 Gymnosperms
11.2.3.1 Monoecious Gymnosperms
Most gymnosperms are monoecious (Givnish 1980). There
is a striking correlation between the breeding system and the
mechanism of seed dispersal. Almost all gymnosperms are
wind pollinated, except cycads, which are beetle-pollinated.
Monoecious gymnosperms also rely on wind to disperse
their seeds. In contrast, most dioecious gymnosperms pos-
sess fleshy fruits and their seeds are dispersed by animals
(Givnish 1980). Exceptions to this rule have been reported;
for example, trioecious and dioecious populations occur in
Pinus edulis (Floyd 1983).
11.2.3.2 Dioecious Gymnosperms
Phylogenetic analyses indicate that dioecy may have been the
ancestral condition in gymnosperms (Mathews et al. 2010).
In general though, relatively little attention has been paid
to the study of sex determination in dioecious gymnosperms.
The main reason for this is likely due to long generation times
and/or the large size of both plants and their genomes, which
complicate genetic studies (see Leitch and Leitch 2013, this
volume). However, a few species have attracted the attention
of researchers because of their phylogenetic significance
(see also Sect. 14.5 in Murray 2013, this volume).
Ginkgo
The staminate and ovulate trees of Ginkgo biloba possess the
same number of chromosomes (2n ¼ 24) and share almost
identical chromosome morphology. The only difference is
that in the ovulate trees; four chromosomes of the somatic
complement have satellites while in the staminate tree only
three chromosomes have satellites. In the male plant, the pair
of short sub-telocentric chromosomes, only one of which
has a satellite, is believed to be the sex chromosomes.
An XY type of sex determination is assumed since the
male possesses a heteromorphic pair of chromosomes (Lee
1954). To establish the necessary molecular tools to under-
stand the evolution of seeds and pollen, Brenner et al. (2005)
created a cDNA library and an EST dataset from the repro-
ductive structures of male (microsporangiate), female
(megasporangiate), and vegetative organs (leaves) of Ginkgo
biloba. The analysis of this EST database from G. biloba has
revealed genes that are potentially unique to gymnosperms.
Many of these genes display a degree of homology with fully
sequenced clones from the ESTs of a cycad. Other Ginkgo
ESTs were found to be similar to developmental regulators
in higher plants. This work has set the stage for future studies
11 Chromosomes and Sex Differentiation
on Ginkgo as a means to better understand seed and
pollen evolution, and resolve the ambiguous phylogenetic
relationship of G. biloba to other gymnosperms.
Cycads
In Cycas pectinata, the male and female plants have the
same number of chromosomes (2n ¼ 22) with almost iden-
tical chromosome morphology. The only difference is that in
the female plant two chromosomes of the somatic comple-
ment (pair III) have satellites, while in the male the same
pair is heteromorphic and only one of its members has a
satellite. This distinction becomes clearly visible when the
two types of haploid complements are observed in pollen
mitosis: one type possesses a satellite chromosome, and the
other does not (Abraham and Mathew 1962). The construc-
tion of a cDNA library and an extensive EST study has
already been started in C. rumphii (Brenner et al. 2003a).
11.2.4 Angiosperm Plants
The ancestral traits of angiosperm flowers are not yet clear;
several hypotheses attempt to explain the origin of the her-
maphrodite flower, which is currently the most wide spread
flower type. According to the “Out Of Male (OOM)/Out Of
Female (OOF) hypotheses,” hermaphrodite flowers developed
as a modification of strobili containing both male and female
flowers (Theissen and Becker 2004). According to the “Mostly
Male (MM) theory,” the hermaphrodite flower developed
from the male flowers of a gymnosperm ancestor by ectopic
formation of ovules (Frohlich and Parker 2000). Different
hypotheses for the origin of the angiosperm hermaphrodite
flower make different predictions concerning the overlap
between the genes expressed in the male and female cones of
gymnosperms and the genes expressed in the hermaphrodite
flowers of angiosperms. The Mostly Male theory predicts that,
of genes expressed primarily in male versus female gymno-
sperm cones, an excess of male orthologs will be expressed in
flowers, excluding ovules, while Out Of Male and Out
Of Female theories predict no such excess. Data obtained by
Tavares et al. (2010) fit better with the Out Of Male and Out of
Female theories. However, it is doubtful that female and male
ancestral characteristics of the angiosperm flower can be
inferred from the number of expressed genes shared by female
and male tissues, as differences between sexes are due to only
a few genes or are quantitative (Tavares et al. 2010).
11.2.4.1 Monoecious Angiosperm Plants
Cucumis melo and C. sativus
Important data concerning the possible mechanisms of sex
determination in Cucurbitaceae have been obtained in melons
(Cucumis melo). In this mostly monoecious species, sex
determination is governed by the genes andromonoecious
171
(a) and gynoecious (g). The dominant allele of the a locus
(CmACS-7 gene; 1-aminocyclopropane-1-carboxylic acid
synthase) results in an arrest of stamen development
(Boualem et al. 2008), while the dominant allele of the g
locus causes an arrest of gynoecium development. Monoe-
cious (A-G-) plants bear male flowers on the main stem and
andromonoecious (aaG-) plants bear female or hermaphrodite
flowers on the axillary branches. Gynoecious (AAgg) and
hermaphrodite individuals (aagg) bear only female or her-
maphrodite flowers respectively. The insertion of the Gyno-
hAT transposon in the proximity of the g gene (CmWIP1) was
shown to be a cause of the gynoecious phenotype of several
lines (G to g change by hypermethylation of the promoter of
the g gene, i.e., CmWIP1). The occasional presence of flowers
with stamens and reduced ovaries suggests that DNA
hypermethylation of CmWIP1 can be reduced during somatic
development of gynoecious plants (Martin et al. 2009). Sur-
prisingly, both CmACS-7 and its homolog from C. sativus are
specifically expressed in female buds. The role of
1-aminocyclopropane-1-carboxylic acid synthase in anther
arrest seems to be indirect and inter-organ communication is
probably responsible for anther arrest (Boualem et al. 2009).
An analysis of the whole genome sequence of C. sativus
revealed that the evolution of unisexual flowers in cucurbits
may have involved the acquisition of two ethylene-
responsive elements (AWTTCAAA) and one flower meri-
stem identity gene LEAFY-responsive element (CCAATGT)
of the ACS genes (Huang et al. 2009). Extensive EST analy-
sis in unisexual and bisexual flower buds (using 454
sequencing) showed that six auxin-related genes (auxin can
regulate sex expression by stimulating ethylene production)
and three short-chain dehydrogenase or reductase genes
(homologs to the sex determination gene ts2 in maize) are
more highly expressed in unisexual flowers then in hermaph-
rodite flowers (Huang et al. 2009).
Zea mays
The formation of unisexual flowers in maize requires the
selective elimination and sexual maturation of floral organs
in an initially bisexual floral meristem. Elimination of pistil
primordia occurs in the primary and secondary florets of
tassel spikelets and in the secondary florets of ear spikelets.
Ill-fated pistil cells undergo a cell death process associated
with nuclear degeneration in a specific spatial-temporal pat-
tern that begins in the subepidermis, eventually aborting the
entire organ. The sex determination genes tasselseed1 (ts1)
and tasselseed2 (ts2) are required for death of pistil cells,
and ts1 is required for the accumulation of ts2 mRNA in
pistil cells. All pistil primordia express ts2 RNA but func-
tional pistils found in ear spikelets are protected from cell
death by the action of the silkless1 (sk1) gene, which blocks
tasselseed-induced cell death in the pistil primordia of pri-
mary ear florets. This basic model for the control of pistil
172
fate by the action of the ts1-ts2-sk1 pathway was proposed
by Calderon-Urrea and Dellaporta (1999). TASSELSEED2
converts steroids with specificities found at positions 3 and
17, and several dicarbonyl and quinone compounds,
thus establishing TASSELSEED2 as a plant 3beta/17beta-
hydroxysteroid dehydrogenase and carbonyl/quinone reduc-
tase. Taken together, the genetic data and the substrate
specificities determined suggest that TS2 converts specific
plant compounds and acts as a pre-receptor control mecha-
nism in a manner similar to that of mammalian hydroxy-
steroid dehydrogenases (Wu et al. 2007). Later, studies by
Parkinson et al. (2007) identified the role of two other genes
in maize sex determination. Genes required to maintain
repression (rm6) and mediator of paramutation 1 (mop1;
putative RNA-dependent RNA polymerase) are involved in
the suppression of the expression of silkless in the male
inflorescence. Rmr6 maintains maize’s monoecious pattern
of sex determination by restricting the function of the pistil-
protecting factor SILKLESS1 from the apical inflorescence
(Parkinson et al. 2007). The exact mechanism leading
from TASSELSEED2 action to proper tassel formation is
still unknown. It is, however, known that further genes
are involved in stamen development promotion and gynoe-
cium suppression: indeterminate spikelet1 (known also as
Tasselseed6) genes and the group of Squamosa-promoter
Binding Protein (SBP) box containing genes. Intron-exon
structures as well as phylogenetic data support the division
of these family members into six groups. The SBP-box
genes upregulated in feminized tassels fall into two groups
(out of six groups of SBP-box genes that have been distin-
guished in Zea mays according to phylogenetic analysis) that
share common structural motifs and include the presence of
a target site for miR156. Small RNA blots showed that
miR156 levels are decreased in both mop1 and ts1 mutants.
While there is a correlation between miR156 levels and
SBP-box gene transcript levels, this correlation is not abso-
lute, and thus it is hypothesized that decreased levels of
miR156 may provide competency for SBP-box gene
upregulation by other common factors yet to be identified.
A model that suggests a putative link between ts1, ts2, ts4,
Ts6, and mop1 in the sex-determination pathway was put
forward by Hultquist and Dorweiler (2008). Progress has
also been made in the study of the molecular mechanism
of action of the putative master gene of sex determination in
maize.
Acosta et al. (2009) positionally cloned and charact-
erized the function of the sex determination gene ts1. The
TS1 protein encodes a plastid-targeted lipoxygenase with
predicted 13-lipoxygenase specificity, which suggests that
TS1 may be involved in the biosynthesis of the plant hor-
mone jasmonic acid. In the absence of a functional ts1 gene,
lipoxygenase activity was missing and endogenous
B. Janoušek et al.
jasmonic acid concentrations were reduced in developing
inflorescences. Application of jasmonic acid to developing
inflorescences rescued stamen development in mutant
ts1 and ts2 inflorescences, revealing a role for jasmonic
acid in male flower development in maize (Acosta et al.
2009; Browse 2009). This suggests that jasmonic acid is
likely to be involved in more complex effects than just the
control of ts2. An updated model of sex determination in
Zea is summarized in Fig. 11.1, which represents
improvements to the original scheme suggested by Hultquist
and Dorweiler (2008).
11.2.4.2 Dioecious Angiosperm Plants
Genus Silene
Silene latifolia: A Model for the Study of Sex Determination
Silene latifolia (Fig. 11.2a, b) is probably the best-studied
plant sexual model. Chromosomes of S. latifolia (formerly
Melandrium album) were first described in 1923 (Blackburn
1923; Winge 1923). Shortly after the species was used by
Correns to study sex ratio bias (reviewed in Correns 1928a).
The nuclear genomes of S. latifolia and S. dioica are relatively
large and arranged into 12 chromosome pairs (see
Fig. 11.3a–d). The pair of sex chromosomes is the largest in
the genome: the Y is 1.4 times longer than the X chromosome.
Deletion mutants have been an important tool in studies of sex
determination in S. latifolia. For example, the use of deletion
mutants has enabled the physical mapping and functional
characterization of regions of interest within the sex
chromosomes. Historically, spontaneous aberrant Y
chromosomes were first studied by Westergaard (1946). By
analysing different types of aberrations, he was able to con-
clude that in S. latifolia three different regions within the Y
chromosome were important for correct sex expression in
males. The upper region of the q-arm is critical for the
suppression of gynoecium formation, the medium region of
the Y is necessary for male promotion, and a portion of the
q-arm is needed for male fertility (Westergaard 1946).
The default sex in S. latifolia is female: when the Y is
completely missing, flowers form only pistils. Recently,
large scale deletions (disruptions) of the Y chromosome
were introduced by means of either X-ray or gamma-
irradiation. Lebel-Hardenack et al. (2002) irradiated pollen
grains using a Siefert X-ray machine, while Farbos et al.
(1999) used 60Co as a source of gamma rays. These studies,
in addition to the deletion mutants observed already by
Westergaard (1946), revealed hermaphrodites and infertile
males. This can be taken as confirmation of Westergaard’s
model of the Y chromosome in S. latifolia. The model of
Y chromosome organization was improved by Zluvova et al.
(2007), who showed the existence of male fertility genes
close to the stamen promoter.
11 Chromosomes and Sex Differentiation
Fig. 11.1 Sex determination in maize (Zea mays). This scheme
represents an updated “tasselseed based” control pathway of sex deter-
mination in maize. Only the variant that occurs in tassel is displayed.
The genes that push development in the male direction are written in
plain text whereas genes pushing development in the female direction
are underlined. The names of gene products and names of mutants that
led to gene identification are in brackets. Broken lines (e.g., from
At present, few experimental data are available concerning
the mechanism of gynoecium suppression in males of
S. latifolia. Histological studies show a reduction of cell
division in the central part of the male flower meristem
(Matsunaga et al. 2004) while molecular studies have revealed
the role of homologs of Arabidopsis thaliana SHOOTMER-
ISTEMLESS (STM) and CUP SHAPED COTYLEDON (CUC)
1 and CUC2 genes in the arrest of gynoecium development in
S. latifolia males (Zluvova et al. 2006). The data of Matsunaga
et al. (2004) and Zluvova et al. (2006) suggest that the absence
of STM and the presence of CUC 1 and CUC2 transcripts in the
central part of the male flower meristem are the cause of
reduced meristematic activity in this region. Independent of
this pathway, gynoecium development in S. latifolia is also
173
silkless1) denote genes not active in male inflorescences. The box
symbolizes that the control pathway from tasselseed2 to pistil abortion
and stamen promotion remains mostly unknown, though some genes
have been identified. The dotted line symbolizes an artificial treatment.
As apparent from the scheme, the role of jasmonate is probably more
diverse than simply controlling tasselseed2 expression (modified
according to Hultquist and Dorweiler 2008)
suppressed by the action of the CLAVATA1 gene, a putative
member of the CLAVATA-WUSCHEL pathway (Koizumi
et al. 2010). The results of Kazama et al. (2009) also indicate
a possible role of SUPERMAN-like gene in the suppression of
anther development in S. latifolia females.
Silene latifolia: A Model for the Study of Evolutionary
Dynamics of Sex Chromosomes
While the majority of findings concerning sex chromosome
evolution have come from research performed in human
and animal models, a few plant species appear to be more
suitable models for sex chromosome evolution owing to
their recently evolved sex chromosomes (Table 11.1).
Sex chromosomes, along with B chromosomes, are amongst
174
Fig. 11.2 Examples of model dioecious flowering plants. (a) Silene
latifolia female flower, (b) S. latifolia male flower, (c) S. colpophylla
female flower, (d) S. colpophylla male flower, (e) Rumex acetosa
female flower bud section, (f) R. acetosa male flower bud section.
Bars represent 5 mm (a–d) or 5 mm (e–f)
the few parts of a plant genome that are feasible for chromo-
some painting techniques. While in animals, ordinary
autosomes have specific patterns of DNA organization that
allow researchers to distinguish individual chromosomes
using complex probes, plant genomes are relatively homog-
enous and generally have few chromosome-specific DNA
structures and sequences. In S. latifolia, microdissected sex
chromosomes were used by Scutt et al. (1997) to investigate
their genomic organization. The experiment was based on a
semi-automatic technique of ablation of chromosomes and
the mechanical transfer of chromosomes of interest to a
membrane (polyester disk). Both X and Y chromosomes
were used for DOP-PCR amplification and PCR products
were subsequently labelled for FISH experiments (DOP-
PCR is the method of choice for amplification of anonymous
DNA with partially degenerated primers (Telenius et al.
1992)). Although sex chromosomes in S. latifolia are signif-
icantly different in their structure, the use of both probes
(X- and Y-derived) revealed no sex chromosome-specific
signal pattern. Similar data were obtained from experiments
by Matsunaga et al. (1999).
A novel approach, which combined microdissection
techniques and FISH procedures, was used by Hobza et al.
(2004). To avoid any impurities during the dissection
B. Janoušek et al.
Fig. 11.3 Examples of chromosome preparations of dioecious species
with heteromorphic sex chromosomes. (a) Silene latifolia, female meta-
phase, (b) S. latifolia, male metaphase, (c) S. dioica, female metaphase,
(d) S. dioica, male metaphase, (e) Rumex acetosa, female metaphase,
(f) R. acetosa, male metaphase. The bar represents 5 mm
process, all manipulations were carried out with a laser
beam using the PALM MicroLaser system (Fig. 11.4). The
hybridization procedure was significantly shortened (1 h),
and the amount of probe was decreased to a low concentra-
tion (30 ng/slide). Specific signals were observed using both
X and Y probes. The differential labelling patterns of
S. latifolia sex chromosomes under specific FISH conditions
show rapid evolution of repetitive elements in the early
stages of sex chromosome divergence (Hobza et al. 2004).
Manual dissection of sex chromosomes was also success-
fully applied in experiments by Delichere et al. (1999),
leading to the discovery of the first active gene to be isolated
from a plant Y chromosome. Hernould et al. (1997) also
performed microdissection, using an inverted microscope
with extended microneedles operated by an electric micro-
manipulator. The glass tips of microneedles carrying chro-
mosome fragments were broken off and pooled in an
Eppendorf tube for subsequent experiments. Delichere
et al. (1999) used 10 microdissected Y chromosomes as a
template for DOP-PCR. The amplified DNA was used as a
probe for screening a premeiotic male flower cDNA library
to select Y-linked expressed sequences. To verify Y-linkage,
segregation analysis was performed. Individual Y
chromosome-derived clones were hybridized with restricted
11 Chromosomes and Sex Differentiation 175

Table 11.1 Comparison of characteristic properties of the most studied plant dioecious models (chrs ¼ chromosomes, F ¼ female, M ¼ male,
My ¼ million years)
Carica papaya L. Silene latifolia Poiret. Rumex acetosa L.
Common name Papaya White campion Sorrel
Family Caricaceae Caryophyllaceae Polygonaceae
Genome size 1C ¼ 0.372 pg 1C ¼ 2.86 pg 1C ¼ 3.55 pg
Karyotype 2n ¼ 18 sex chromosomes 2n ¼ 24 (11pairs of autosomes plus 1 2n ¼ 14(15) (6 pairs of autosomes plus XX
homomorphic pair of sex chromosomes) or XY1Y2)
Heterogametic Male Male Male
sex
Sex chrs (F/M) Not detected XX/XY (Blackburn 1923) XX/XY1Y2 (Kihara and Ono 1923)
Size of sex chrs Not detected The largest Y (9 %), the second X (8 %), The largest X (13 %), the second Y1 + Y2
smaller autosomes (25 %), small autosomes
Chromatin of Non-recombining region of the Y Standard as in autosomes (Grant et al. 1994) The Ys are heterochromatic, histone H4-
sex chrs heterochromatic (Zhang et al. 2008) underacetylated (Lengerova and Vyskot
2001)
Age of sex chrs 2–3 My (Zhang et al. 2008) Less than 5–10 My (Filatov 2005) 15–20 My (Jamilena et al. 2008)
Type of sex Dominant Y in males (mammalian Dominant Y in males (mammalian type) X/A ratio (F ¼ 1, M ¼ 0.5) (Drosophila
determination type) type)
Accumulation In the non-recombining region of Plastid DNA, DNA repeats, Y-specific repeats, microsatellites (Jamilena
of Y-repeats the Y retrotransposons, microsatellites et al. 2008)
(Kejnovsky et al. 2009)
Availability of Full genome sequence available At least 10 X (and) Y linked publically No sex chromosome linked genes available
X-genes (Liu et al. 2004) available genes yet

In spite of their relatively young age it was shown that

Fig. 11.4 Laser microdissection on Silene latifolia and Rumex acetosa
metaphase chromosomes. The S. latifolia Y chromosome is localized
under the inverted microscope (a). The membrane is cut around the
selected chromosome using a laser microbeam, and the chromosome Y
(b) and X (c) is transferred into the cap of a PCR tube. Similarly, the
R. acetosa X chromosome is selected and separated from the rest of the
chromosomes by microdissection (d–f)
genomic DNA from male and female parental plants as well
as their progeny. Out of the 115 clones selected after
hybridization of complex Y probes with the cDNA library,
only five clones revealed sex linkage after segregation
experiments. A later study reported a level of DNA poly-
morphism in the Y-linked copy of the gene SlX1/SlY1 that
was twenty times lower than the X-linked copy. These data
were the first to suggest that processes involved in Y chro-
mosome degeneration were also acting in the relatively
young chromosomes of S. latifolia (Filatov et al. 2000).
there is a similar gradient in silent site divergence between
the X and Y copies of sex-linked genes on the sex
chromosomes S. latifolia (Nicolas et al. 2005; Bergero
et al. 2007). Silene latifolia Y-linked genes tend to evolve
faster at the protein level than their X-linked homologs, and
they have lower expression levels. Analysis of several
Y-linked gene introns suggest they act as sites for
transposable-element accumulation, which likely accounts
for their increased length (Marais et al. 2008). These signs
of degeneration are similar to those observed in animal
Y-linked and neo-Y chromosome genes. Cermak et al.
(2008) carried out a global survey of all of the major types
of transposable elements in Silene latifolia. The localization
of elements by FISH revealed that most of the Copia
elements had accumulated on the Y chromosome. Surpris-
ingly, one type of Gypsy element, which was similar to the
Ogre elements known from legumes, was almost absent on
the Y chromosome but otherwise uniformly distributed on
all chromosomes. Other types of elements were ubiquitous
on all chromosomes. Moreover, Cermak et al. (2008)
isolated and characterized two new tandem repeats. One of
them, STAR-C, was localized at the centromeres of all
chromosomes except the Y chromosome, where it was pres-
ent on the p-arm. Its variant, STAR-Y, which carries a small
deletion, was specifically localized on the q-arm of the Y
chromosome. FISH analysis of other Silene species revealed
that some elements (e.g., Ogre-like elements) are confined to
176
the section Elisanthe while others (e.g., Copia or Athila-like
elements) are also present in more distantly related species.
The unique pattern of repeat distribution found on the Y
chromosome, where some elements have accumulated
while other elements are conspicuously absent, probably
reflects the different forces shaping the evolution of the
Y chromosome (Cermak et al. 2008). Kubat et al. (2008)
have shown that microsatellites accumulate in the q-arm of
the Y chromosome (the arm containing the pseudoautosomal
region) and this agrees with the findings of other authors
(Morgante et al. 2002) showing that microsatellites are pref-
erentially located in non-repetitive sequences.
However, repetitive sequences are not the only types of
DNA to accumulate in the non-recombinant part of plant Y
chromosomes. Sequence analysis has revealed that one
of the Y chromosome-derived BACs contains part of the
plastid genome, indicating that these plastid sequences have
been transferred to the Y chromosome and may also contrib-
ute to its large size. Kejnovsky et al. (2006) found that
plastid sequences located on the Y chromosome had higher
rates of divergence in non-genic regions than in genic
regions, which showed only very low (max 0.9 %) diver-
gence from their plastid homologs.
The study of the Y chromosome-derived library has also
revealed a case of horizontal gene transfer of a DNA frag-
ment from the bacterium—Ralstonia solanacearum to the
genome of S. latifolia. The homologs of this fragment
(MK14) contain sequences that show similarities to a gene
coding bacterial sulphate adenylyltransferase (CysN) and
to a gene encoding uroporphyrin-III C-methyltransferase
(nirE) (Talianova 2009). The reaction catalysed by
sulphate adenylyltransferase constitutes the first enzymatic
step in sulphate utilization following the uptake of sulphate
(Leyh et al. 1992). Uroporphyrin-III C-methyltransferase in
bacteria is involved in the biosynthesis of corrinoids such as
vitamin B12, sirohaem and coenzyme F430 (Vévodová et al.
2004). The homologs of this fragment were subsequently
found also in species that do not possess sex chromosomes,
suggesting that the Y chromosome is unlikely to be more
prone to the accumulation of horizontally acquired genes
than regular autosomes.
Other Silene Species
Silene is a large genus, where the majority of species possess
12 pairs of chromosomes. In some dioecious species there is
one pair of sex chromosomes plus 11 pairs of autosomes.
This pattern which is supported by molecular data (Nicolas
et al. 2005; Bergero et al. 2007), suggests that one pair of
autosomes evolved into the pair of sex chromosomes in
subgenus Behenantha, section Melandrium (classification
according to Rautenberg et al. 2010). In contrast, the origin
of sex chromosomes in subgenus Silene (classification
according to Eggens et al. 2007, and Rautenberg et al.
2010) is probably different as indicated by the phylogenetic
B. Janoušek et al.
and genetic mapping data in S. colpophylla. Though many
species share the same number of chromosomes, there are
big differences in genome size among Silene species. For
example, S. vulgaris and S. pendula belong to a group of
small-genomed species (about 2 pg/C), while S. latifolia and
S. chalcedonica possess relatively large genomes (3–5 pg/C,
respectively). These differences raise questions about the
mechanisms that have contributed to genome size evolution.
Recent data indicate that there are large blocks of
subtelomeric heterochromatin missing in some of the
smaller genomes, which may account for much of these
differences (Cermak et al. 2008). Subgenus Behenantha
contains only five dioecious species: S. latifolia, S. dioica,
S. diclinis, S. marizii, and S. heuffelii. Subgenus Silene
contains several dioecious and subdioecious species, the
best studied being S. otites and S. colpophylla (Fig. 11.2c,
d) of the former section Otites, Wrigley (1986), now subsec-
tion Otites of the section Siphonomorpha, according to
Oxelman (2010). Dioecy in section Melandrium is of mono-
phyletic origin: the same pair of autosomes evolved into sex
chromosomes in all species. In section Otites, genetic and
phylogenetic analyses have been conducted to understand
the sex-determining system and the origin of sex
chromosomes in S. colpophylla (Mrackova et al. 2008).
Here, it was shown that genes that are sex-linked in S.
latifolia are also linked to each other in S. colpophylla, but
they are not sex-linked. This finding demonstrates that the
sex chromosomes in S. colpophylla (which are homomor-
phic) evolved from a different pair of autosomes than in S.
latifolia. Phylogenetic analyses also support the view that
the sex determination system of S. colpophylla, although it is
XX/XY (similarly to section Melandrium), evolved indepen-
dently in section Otites (Mrackova et al. 2008).
Genus Rumex
Various types of reproductive systems occur in Rumex:
hermaphroditism, polygamy, gynodioecy, monoecy and
dioecy (reviewed in Navajas-Pérez et al. 2005). In dioe-
cious Rumex species, two different sex-chromosomal
systems and sex-determining mechanisms have been
described: XX/XY with an active Y chromosome (e.g.,
Rumex acetosella) and XX/XY1Y2 with sex determination
based on the X/A ratio (e.g., Rumex acetosa, Figs. 11.2e, f
and 11.3e, f). There is one exceptional species, Rumex
hastatulus, which has two chromosomal “races”: the
Texas race possessing XX/XY system and the North
Carolina race with an XX/X Y1Y2 system. In this species,
the X/A ratio controls sex determination, but the presence
of the Y chromosome is necessary for male fertility (Smith
1963). In R. acetosa repetitive sequence similarity between
both Y chromosomes suggests that they probably originated
from one Y chromosome that underwent centromere
fission and gave rise to a pair of metacentric chromosomes
possessing identical chromosomal arms (isochromosomes).
11 Chromosomes and Sex Differentiation
These isochromosomes were subsequently modified by
deletions (Rejon et al. 1994). A recent phylogenetic study
(Navajas-Pérez et al. 2005) indicates that all dioecious
Rumex species evolved from a common hermaphroditic
ancestor. The switch from a sex-determining mechanism
based on the active role of the Y chromosome to a mecha-
nism based on the X/A ratio occurred at least twice
(Navajas-Pérez et al. 2005). The role of the X/A ratio in
the sex determination of R. acetosa (reviewed by Parker and
Clark 1991) resembles the sex-determining system of Dro-
sophila, where the primary genetic sex-determining signal is
provided by the ratio of X-linked genes to autosomal genes
(Pomiankowski et al. 2004).
Hemp (Cannabis sativa)
Cannabis sativa or hemp is one of the few dioecious plant
species possessing heteromorphic sex chromosomes, albeit
the size difference between the X and Y chromosome is
small (reviewed in Peil et al. 2003). Males are heterogametic
(XY) and females homogametic (XX). The sex-determining
system is the Drosophila type: the ratio of Xs to autosomes
determines the sex rather than the active Y chromosome
(revieved by Parker and Clark 1991). In contrast to other
dioecious models (e.g., Silene latifolia and Rumex acetosa)
the Y chromosome of C. sativa probably does not contain the
genes necessary for male fertility. After stamen induction by
ethylene synthesis inhibiting drugs (silver nitrate and silver
thiosulphate anionic complex), fertile stamens develop in
genetically female plants of hemp (Mohan Ram and Sett
1982). Sex determination in hemp is therefore probably
under control of plant hormones, as changes in their levels
often lead to sex changes (Chailakhyan and Khryanin 1978,
1979). Some features of Y chromosome degeneration have
been found in hemp, for example, an accumulation of LINE-
like retrotransposons at the terminal region of the longer arm
of the Y chromosome has been reported (Sakamoto et al.
2000). Several genes that are involved in sex determination
and/or sexual dimorphism have been identified using cDNA
AFLP (Moliterni et al. 2004).
Hop (Humulus lupulus)
In spite of its agronomical importance, relatively little is
known about the sex determination mechanism in hops. It is
known that hops possess heteromorphic sex chromosomes
and sex determination of XX/XY1Y2 i.e., sex determination
is based on the ratio of the dosage of X chromosomes and
autosomes (Parker and Clark 1991). Shephard et al. (2000)
showed that obvious sex-related differences were already
present in the plant at the stage of initiation of inflorescences
suggesting that the genes involved in sex determination are
probably already at work in advance of floral organogenesis
(Shephard et al. 2000). Numerous sex-specific markers have
177
been isolated in H. lupulus (Jakse et al. 2008) and a linkage
map is available together with cytogenetic methods to distin-
guish between hop chromosomes (Karlov et al. 2003).
Papaya (Carica papaya)
Papaya is a favorite dioecious model because of its small
nuclear genome size and importance as a crop. Papaya is
polygamic and forms three basic sexual types: males,
females, and hermaphrodites. Sex in papaya is determined
by a single locus, which may have three different alleles
(M1—male, M2—hermaphrodite, and m—female) (Storey
1969). Males must have the M1 dominant allele, while
hermaphrodites possess the M2 allele and mm forms
females. The combination of any two dominant alleles
leads to seed abortion. Since males are heterogametic and
females are homogametic, the sex determination can be
classified as the XY type with a dominant Y chromosome.
A large amount of DNA polymorphisms near the sex locus
led to the recent discovery of the Y chromosome in papaya
(Ma et al. 2004). There is a small 4–5 Mb region, called the
MSY (male specific region Y), which harbors the primary
sex-determining genes. Yu et al. (2007) proposed the
hypothesis that recombination suppression in the sex-
determining region of the Y chromosome is the result of its
close proximity to the centromere.
Meadow-rue (Thalictrum dioicum)
Meadow-rue is a common weed in the Ranunculaceae fam-
ily. The unisexual flowers lack any rudiments of the opposite
sex since developmental arrest occurs very early during floral
ontogeny. Thalictrum dioicum has homomorphic sex
chromosomes and sex is genetically determined by a few
loci. Males are heterogametic, and the genetic system of
sex determination looks like the dominant Y system that is
present in Silene latifolia and humans. The Y chromosomes
in dioecious species of Thalictrum are probably not largely
degenerate since YY males are viable even when no X copy
is present in T. fendleri (Kuhn 1939). In dioecious plants like
T. dioicum and Spinacia oleracea (see below), where
vestiges of the opposite sex organs are missing, homeotic
MADS-box genes are good candidates for sex determination
genes. In T. dioicum, Di Stilio et al. (2005) isolated the organ
identity genes (Agamous, Apetala3, and Pistillata) and
showed that they had been duplicated and had diverged in
expression. Efficient virus-induced gene silencing (VIGS),
using tobacco rattle virus (TRV) vectors, has been achieved,
permitting deep analysis of sex determination mechanisms
(Di Stilio et al. 2010).
Spinach (Spinacia oleracea)
Spinach is predominantly a dioecious species. However,
genetically-determined monoecious lines also exist (Janick
178
and Stevenson 1954), and sex expression also depends on
environmental influences. For example, high temperatures
promote maleness in monoecious lines (reviewed in Iizuka
and Janick 1962). The sex-determining system is XX/XY
(reviewed in Haga 1934). X and Y chromosomes are of
similar size and morphology, but some Y chromosomes
lack a 45S rDNA locus (Lan et al. 2006). Sex determination
in S. oleracea is based on a single locus with three alleles: X,
Xm and Y (Janick and Stevenson 1954). The allele Xm is
incompletely dominant because XmXm individuals are mon-
oecious, but individuals with XmX produce a higher propor-
tion of pistillate flowers. The Y allele is completely
dominant, and thus XY and XmY plants show the male
phenotype.
The exact molecular basis of the sex determination sys-
tem in S. oleracea is unknown. However, a study of the B
class floral identity genes SpAPETALA3 and SpPISTILLATA
in S. oleracea showed their effect on sexual dimorphism and
suggested a role in sex determination (Pfent et al. 2005). The
gene SpAgamous is involved in sex-specific gynoecium con-
trol in spinach (Sather et al. 2005). Interestingly, sexual
dimorphism in this species can vary in intensity depending
on the presence of a special locus located on the Y chromo-
some that causes a “bracted male” phenotype showing a
habit different from female plants (reviewed in Iizuka and
Janick 1962). Indeed, there are many indications that the sex
chromosomes of S. oleracea are young, but this does not
necessarily imply that the sex determination system is also
young and it is possible that the sex determination system is
derived from monoecy. In this case, a single gene controlling
the number and/or position of male and female flowers could
have been directly transformed into a sex determining gene
by sexually antagonistic selection. Given the small size of
the spinach genome, it is suitable for basic genomic studies
(Khattak et al. 2006) and recent RNAi technology applying a
VIGS approach has permitted a functional genomic strategy
to be adopted. Such studies showed how the suppression of
B class genes led to sex reversal via a homeotic transforma-
tion of stamens into carpels (Sather et al. 2010) confirming
the view that sex determination in spinach is based on the
regulation of B class gene expression.
Mercurialis annua
Mercurialis annua has a simple genetically-based sex deter-
mining system. There seem to be three unlinked loci—A/a,
B1/b1, and B2/b2—which act complementarily. The male
plants usually need the dominant A allele and at least one
dominant B allele. Moreover, in Mercurialis sex can be
modulated by exogenously applied growth regulators. In
females, the presence of trans-zeatin was shown. An appli-
cation of auxin leads to masculinisation of female flowers
(Hamdi et al. 1987). Khadka et al. (2002) reported the
characterization of a previously identified male-specific
B. Janoušek et al.
DNA marker, OPB01-1562, from the diploid dioecious
M. annua. RNA blot hybridization with OPB01-1562 and
MARL-1 detected a transcript that was expressed strongly in
the stems and flowers of females but not males. The
M. annua female-expressed (Mafex) transcript may thus
play a role in sex determination (Khadka et al. 2005).
Hybridization and polyploidy are widely believed to be
important sources of evolutionary novelty in plant evolution
(see for review, Soltis et al. 2010; Fawcett et al. 2013, this
volume). Both can lead to novel gene combinations and/or
novel patterns of gene expression, which in turn provide the
variation on which natural selection can act. Obbard et al.
(2006) used nuclear and plastid gene trees, in conjunction
with morphological data and genome size measurements, to
show that both processes have been important in shaping the
evolution of Mercurialis. Their results indicate that hexa-
ploid populations of M. annua, in which the otherwise
rare sexual system androdioecy is common (the occurrence
of males and hermaphrodites), is of allopolyploid origin
involving hybridization between a monoecious autotetra-
ploid lineage of M. annua and the related dioecious diploid
species M. huetii—an event that brought together the genes
for specialist males with those for hermaphrodites (Obbard
et al. 2006). Dorken and Pannell (2008) found that the
progeny sex ratio is strongly dependent on density, with
fewer males produced when plants are grown at low density.
This occurred in part because of a flexible adjustment in
pollen production by hermaphrodites, which produced more
pollen when grown at low density than at high density. These
results provide support for the prediction that environmental
conditions govern sex ratios through their effects on
the relative fertility of unisexual versus hermaphrodite
individuals (Dorken and Pannell 2008).
Bryonia dioica: The First Dioecious Model in Which
the Genetic Basis of Sex Determination was Elucidated
Most species of the Cucurbitaceae family comprising c. 800
species have unisexual flowers, 460 are monoecious, and 340
are dioecious. Some species produce a mixture of bisexual,
female, and male flowers in various intra- and inter-individual
patterns, and populations can be andromonoecious, androdi-
oecious, gynomonoecious or gynodioecious (Kocyan et al.
2007). Phylogenetic studies suggest that dioecy is the ances-
tral state in this family (Zhang et al. 2006). However, as
various switches to monoecy or to other types of reproductive
systems, including androdioecy, have occurred during the
evolution of Cucurbitaceae, it is therefore difficult to precisely
ascertain how old the sex chromosomes in a given species are
(Renner et al. 2007). The best-studied genus containing dioe-
cious species is Bryonia. Genetic crosses between the dioe-
cious B. dioica and the monoecious B. alba in 1903 provided
the first clear evidence for Mendelian inheritance of sexual
phenotypes (dioecy) and made B. dioica the first organism for
11 Chromosomes and Sex Differentiation
which the XY sex-determination was experimentally proven
(Correns 1928b). Applying molecular tools to this system,
Oyama et al. (2009) showed that the size of the non-recom-
bining region may differ between the north-European and
southern-European populations. Important data concerning
the possible mechanisms of flower sex determination in
monoecious Cucurbitaceae were recently obtained in melons
(C. melo) and in the cucumber (C. sativus, see Sect. 11.2.4.1.1
in this chapter).
Date Palm (Phoenix dactylifera)
The date palm is a dioecious species displaying strong dimor-
phism between pistillate and staminate flowers. Siljak-
Yakovlev et al. (1996) described a cytological method using
chromomycin staining that demonstrates the occurrence of sex
chromosomes based on distinctive nucleolar heterochromatin.
This offers the possibility of identifying male and female
individuals by a simple analysis of root meristems. This obser-
vation has been extended by in situ rDNA hybridization,
confocal microscopy and dual-label flow cytometry of nuclei
(Siljak-Yakovlev et al. 1996). The mechanism of arrest of
gynoecium development in the date palm appears to be similar
to that in Silene latifolia. The earliest sex-related difference in
flower buds is observed when the number of cells in the
gynoecium of pistillate flowers is higher than that in their
staminate counterparts. In the pistillate flower, staminodes
(sterile stamens) display precocious arrest of development
followed by cell differentiation. In the staminate flower,
pistillodes (sterile gynoecium) undergo some degree of differ-
entiation, but their development ceases shortly after the ovule
has been initiated. Staminode and pistillode cells exhibit
nuclear integrity although they do not show any accumulation
of histone H4 gene transcripts. These results suggest that the
developmental arrest of sterile sex organs and the subsequent
unisexuality of date palm flowers result from a cessation of cell
division and precocious cell differentiation rather than from
cell death (Daher et al. 2010).
11.3 Sexual Dimorphism
Sexual dimorphism, the sex-specific expression of some
genes not involved directly in sex determination, presum-
ably results from different modes of selection operating in
males and females: males are limited in their reproductive
success by access to mates, whereas females are more lim-
ited by resources (Bateman 1948; Jones et al. 2002). In
animals, the evolution of sexual dimorphism is primarily
driven by competition between males and selection for traits
recognized by females as indicators of male fitness
(reviewed by Hall et al. 2000). Similar principles may be
at work in animal-pollinated plants. In S. latifolia, odor-
179
compounds involved in pollinator attraction differ signifi-
cantly between sexes, suggesting that selection for higher
attractiveness among competing males is mediated by the
sensory ecology of the pollinator (Waelti et al. 2009). In
addition, males produce on average up to 16 times more
flowers than pollinated females (Laporte and Delph 1996).
This difference in flower number may be driven by a combi-
nation of male competition and, at least partly, by a higher
consumption of resources by developing seeds in pollinated
female flowers, which results in a trade-off between seed
size and flower number. The difference in flower number is
less pronounced in non-pollinated females, which produce
on average only four times fewer flowers than males
(Laporte and Delph 1996; Steven et al. 2007). It also seems
reasonable to expect that differences in the vegetative parts
of plants evolved in concert with the flower types or archi-
tecture of inflorescences carried by the plant. Many
sexually-dimorphic traits could evolve as a consequence of
their correlation with other sexually dimorphic traits and so
they need not be of adaptive value (Dawson and Geber
1999). For instance, correlations between flower size and
the size of the stem leaves have been reported in many
species (reviewed by Dawson and Geber 1999). Variation
in sex-limited genes with pleiotropic effects and/or linkage
between sex-limited loci also occurs in S. latifolia (Steven
et al. 2007). These authors statistically predicted that selec-
tion for increased flower number in males along with weak
selection for increased flower size in females could lead to
dimorphic evolution in several other traits including leaf
mass.
Almost all of the sexually dimorphic traits in S. latifolia
described so far become apparent only after the initiation
of flowering. Notable exceptions to this include: sex-
dimorphism in the long-term survival of buried seeds and
burial induced dormancy in S. latifolia (Purrington and
Schmitt 1995), and sex-dimorphism in emergence time and
time to flowering (Doust et al. 1987; Purrington and Schmitt
1998). Zluvova et al. (2010) provided molecular evidence that
sexually dimorphic gene expression is present in S. latifolia at
the rosette stage, a long time before the initiation of flowering.
Recently, Janoušek and Mrackova (2010) have taken up
the so far neglected question of how a system based on two
separate sex-controlling pathways regulated by genes pres-
ent on the Y chromosome (Charlesworth and Charlesworth
1978) is transformed into a system controlled by the X/A
ratio in some species (as in some species of Rumex).
They suggest the following scenario. In the first step
(Fig. 11.5a), dioecy evolves from gynodioecy via a
gynoecium-suppressing gene in the proximity of the male
fertility controlling gene. This process is promoted by
sexually antagonistic selection. The original theory
(Charlesworth and Charlesworth 1978) supposes just one
180
Fig. 11.5 The evolution of the sex-determining pathways in plants.
(a) The theory of the origin of dioecy via male sterility as suggested by
Charlesworth and Charlesworth (1978). Both the gynoecium suppressor
and the stamen (anther) promoter act independently, but their coordina-
tion is achieved by their proximity on the Y chromosome or by their
location in the non-recombining region of the Y chromosome.
(b) Formation of sex chromosomes. Accumulation of sexually antagonis-
tic genes (SAG) and the reduction of recombination frequency between
gynoecium suppressing and male fertility controlling genes creates sex
chromosomes. For simplicity, only one sexually antagonistic gene is
presented. SAG-F means a sexually antagonistic allele advantageous
for females, and SAG-M means a male advantageous sexually antagonis-
tic allele of the same gene. (c) A sexually antagonistic gene(s)-based
B. Janoušek et al.
switch in the sex-determining pathway. Sexual dimorphism (controlled
by SAG-M) is improved step-by-step and starts to act before the Y-linked
genes involved in female and stamen development. At a certain stage, the
expression of both the gynoecium suppressor and the stamen promoter
becomes sex limited as a consequence of their adaptation to sex specific
expression profiles of other genes. (d) The restructuring of the sex
chromosomes. The gynoecium suppressor and stamen promoting gene
(s) are lost from the Y chromosome and transferred to the autosome(s).
(e) The origin of an X/A based sex-determining system. SAG-M is lost
from the Y chromosome and transferred to an autosome. The X/A ratio
becomes crucial for sex determination as SAG-M pushes development
toward the male direction in contrast to SAG-F that pushes development
toward the female direction
11 Chromosomes and Sex Differentiation
male sterility mutation, implying that the male fertility locus
of the gynodioecious ancestor is identical with the anther-
promoting gene of the resulting dioecious species. The pos-
sibility of a step-wise shift in stamen arrest was discussed by
Zluvova et al. (2005), but does not essentially change the
predictions of the model. In a second step (Fig. 11.5b),
sexually antagonistic selection continues and improves the
linkage of the sex-determining loci. Sex chromosomes that
are created by this process can continue to accumulate sexu-
ally antagonistic alleles (Rice 1984). Even in species that are
at this stage of sex determination, it is possible to find early
expressed sexual dimorphism (e.g., Silene latifolia; Zluvova
et al. 2010).
In the third step (Fig. 11.5c), sex-determining genes start
to accommodate to the sex-specific gene expression patterns
controlled by the sexually antagonistic gene(s) (SAG) and
their expression starts to be controlled by these genes. Even-
tually, the gene(s) that previously controlled sexual dimor-
phism become(s) sex-determining gene(s). It is known that
sexually antagonistic genes evolve fast (reviewed in
Qvarnstr€ om and Bailey 2009) and a good example of this
in plants is the Y-linked genes from S. latifolia. The lack
of a Y chromosome in S. latifolia cannot be completely
compensated for by the presence of the genome of the
related species S. viscosa, and anther defects in hybrids
between S. latifolia and S. viscosa resemble two different
mutants lacking part of the Y chromosome (Zluvova et al.
2005). The active role of the Y chromosome in sex determi-
nation is still preserved because the new sex-determining
locus is still located on the Y chromosome. An important
difference from the previous stages is that connection
between the control of stamen promotion and anther sup-
pression is established. This creates new possibilities for the
evolution of the sex-determining system. In a plant species
possessing this kind of sex determination, a single master
gene mutation should be able to cause a male to female
transformation.
In the fourth step (Fig. 11.5d), two things could have
happened. The original sex-determining loci could be lost
from the Y chromosome by chance or the translocation of
the original sex-determining region to the autosomes could
be supported by Y chromosome degeneration due to the
absence of recombination. Since stamen promotion and
gynoecium suppression are already controlled by the single
controlling pathway, the genotypes possessing a transloca-
tion of these genes to autosomes can be selected for because
they can escape from the process of degeneration. The
change of the position of these genes does not influence the
sexual phenotype, as they have sex-limited expression and
are controlled by the gene derived from the gene that
initially controlled only sexual dimorphism.
In the fifth step (Fig. 11.5e), the secondary sex-
determining locus loses its controlling role as well, and
181
control of sex determination is co-opted by alternative
mechanisms, such as an X/A ratio mechanism. The switch
could be similar to the mechanisms described in fishes and
insects (reviewed by Sch€utt and N€othiger 2000; Volff et al.
2007). One of the genes, “a slave” in the sex-determining
pathway located on the X chromosome, can become “a
master”. Simultaneously, the function of this new master
gene is influenced by genes located on autosomes. An
important prerequisite of this kind of transformation of the
sex-determining system is that stamen promotion and gynoe-
cium suppression are controlled by the same pathway. This
fifth step in the evolution of the sex-determining pathway
may have been reached in hemp (Cannabis), hop (Humulus),
and sorrel (Rumex).
Veltsos et al. (2008) performed computer modelling
experiments in order to explain spread of neo-sex
chromosomes in the grasshopper Podisma pedestris. In this
species, neo-X chromosomes (autosomes fused to original
X) and neo-Y chromosomes (unfused autosome) have
spread and become fixed in a large geographical territory.
Computer modelling experiments showed that the continu-
ous invasion of the neo-Y chromosome (carrying a sexually
antagonistic allele) in the hybrid zone can cause selection in
favour of the neo-X chromosome. Afterwards, also the neo-
Y chromosome that is, in the initial phase, continuously
removed by selection can be selected for as a consequence
of the neo-X accumulation. Interestingly, the same mecha-
nism may be involved in the spread and fixation of the
degenerated Y chromosomes. It is apparent that, in many
plant (e.g., Carica papaya, Silene latifolia, and Asparagus
officinalis, reviewed by Ming et al. 2007) and animal species
(e.g., most mammals, reviewed by Wilhelm et al. 2007) this
evolution is not complete because many species still rely on
an active role of the Y chromosome in sex determination.
Instability of sex expression and/or cytologically homo-
morphic sex chromosomes are sometimes taken as a sign of
the primitive status of the evolution of sex-determining
systems (e.g., Vyskot and Hobza 2004). Data obtained in
the animal models, however, suggest that even very
advanced sex-determining systems (as in mammals) can
show a considerable plasticity of sex expression (e.g.,
Bianchi 2002). Only comparative sequencing and phyloge-
netic analyses can answer the question of the age of sex-
determining pathways.
11.4 The Current Status of Research and
Prospects for the Future
An overview of currently used approaches for studying sex
determination in plants and their inter-connection is
provided in Fig. 11.6. Separation of the chromosome(s) by
microdissection and chromosome sorting is the most
182
Fig. 11.6 A diagrammatic sketch of the methods frequently used for
the isolation and characterization of sex-linked DNA. Dissection of
chromosomes is the method of choice for generating complex probes
for FISH (fluorescence in situ hybridization), constructing chromosome-
specific libraries and as a tool for mapping DNA markers using specific
chromosomes as a template. A subtractive approach (e.g., RDA—
representational difference analysis) is used for the isolation of
sample-specific DNA (e.g., in the comparison of male and female
genomic DNA or the comparison of sex-specific expression patterns).
straightforward technique for isolating chromosome-specific
DNA. The use of these techniques enabled researchers
to isolate the first active plant Y-linked gene (Delichere
et al. 1999). Moreover, the construction and utilization of -
chromosome-specific libraries and the use of sorted chromo-
somes as complex probes has improved our knowledge of
sex chromosomes, mainly in Rumex acetosa and Silene
latifolia. However, only the few species with morphologi-
cally different chromosomes are suitable for dissection
techniques.
High-throughput sequencing methods accelerate whole-
genome sequencing (Tuskan et al. 2006; Ming et al. 2008)
and permit the construction of huge EST and genomic
databases. Comparisons of different pools of sequenced
samples (males versus females, generative tissue versus veg-
etative tissue) may help identify sex-determining genes and to
characterize sex expression pathways (Blavet et al. 2011).
Important questions regarding the basic biological pro-
cesses affecting the evolution of sex chromosomes that
could be answered in the next decade are: How do sex
chromosomes degenerate? Is degeneration of genes in non-
recombining regions really a one-way ticket as it is in
many animal species? Is dosage compensation a general
B. Janoušek et al.
Nowadays, traditional molecular subtractive methods are replaced by
comparisons of different DNA databases. Genetic mapping is still the
basic method for analysis of DNA markers and their linkage to pheno-
typically interesting mutations (AFLP—amplified fragment length
polymorphisms; RAPD—Random Amplification of Polymorphic
DNA). A combination of genetic maps with information on complete
genome sequences is a key approach for the identification and isolation
of novel genes and the deduction of their function
phenomenon for regulating the level of expression in
organisms with degenerating sex chromosomes?
Acknowledgment This research was supported by the Grant Agency
of the Czech Republic (grants P501/10/0102, 522/09/0083, and
521/08/0932).
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 Holocentric Chromosomes
12
Petr Bureš, František Zedek, and Michaela Marková

Contents 12.1 Introduction

12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
12.2 Recognition and Verification of Holocentrism . . . . . . . 187
In contrast to the “normal type” of monocentric mitotic
chromosomes, where spindle attachment is restricted to a sin-
12.3 Occurrence of Holocentric Chromosomes in Plants
gle kinetochore, holocentric1 chromosomes are those in
and Other Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
which spindle microtubules attach along the whole length
12.4 Chromatin Structure of Holocentric Chromosomes . 189 through kinetochores that cover a substantial part of their
12.5 Mitosis in Holocentrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 poleward surfaces during mitosis. In addition, holocentric sis-
12.6 Meiosis in Holocentrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 ter chromatids are interconnected along their whole length
12.6.1 Holokinetic Meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 before anaphase disjunction, unlike monocentric chromatids,
12.6.2 Telokinetic Meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 which cohere only in the pericentromeric area. The morpho-
12.7 Holocentric Karyotypes and Their Evolution . . . . . . . . 195 logical distinctions between monocentric and holocentric
12.7.1 Symploidy and Agmatoploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 chromosomes are associated with differences in chromatin
12.7.2 Polyploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 structure and modified mitosis or meiosis, as well as karyotype
12.7.3 The Enigmatic Case of Luzula: Concerted Fission and
True Polyploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
evolution of the holocentrics themselves. In this chapter, we
will survey these aspects of holocentrism and also discuss some
12.8 How and Why Holocentric Chromosomes Originate 202
hypotheses on the origin of holocentric chromosomes, their
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 patterns of occurrence and methods of verification, particularly
among plants.

12.2 Recognition and Verification

of Holocentrism

Multiple spindle attachment along chromosomes was first

unwittingly observed by pioneers of the study of
chromosomes, such as the Belgian Édouard van Beneden
and the Germans Richard Hertwig and Theodor Boveri, at
the end of the nineteenth century. Using light microscopy
and sophisticated staining techniques, they depicted a dis-
tinct and novel pattern of cell division in their cytogenetic
model, the horse roundworm Ascaris megalocephala (¼
Parascaris univalens; Nematoda, Ascaridida). The first to

1
To avoid confusion with the more narrowly defined term
P. Bureš (*) “holokinetic”, which describes meiotic chromosome behaviour as the
Department of Botany & Zoology, Masaryk University, Kotlářská 2, opposite of telokinetic behaviour, we prefer to use the term
Brno 611 37, Brno 621 00, Czech Republic “holocentric” to indicate the opposite of the monocentric chromosomal
e-mail: bures@sci.muni.cz nature.

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 187

DOI 10.1007/978-3-7091-1160-4_12, # Springer-Verlag Wien 2013
188
Fig. 12.1 Mitosis in holocentric organisms. Sister chromatids are
represented by the same shading, either dark or light grey and both
the kinetochore and microtubules are black. (a) Prophase:
chromosomes after replication. The chromatids are connected length-
wise by cohesins, and kinetochore formation begins (CENH3-bound
domains in black). (b) Metaphase: condensed metaphase chromosomes
congress at the equatorial plate, while kinetochores occupy the whole
poleward sides of the chromosomes. (c) Anaphase: migration of sister
chromatids parallel to the equatorial plate
recognize the peculiarity of holocentric chromosomes was the
American zoologist and cytologist Franz Schrader (1935) in
mealy bugs (Hemiptera). The holocentric chromosomal struc-
ture was confirmed experimentally by Schrader’s wife Sally
and Hans Ris using X-radiation to generate chromosome
fragments that behaved like individual chromosomes and
were consistently transmitted to the next cell generation
(Hughes-Schrader and Ris 1941). To prove the existence of
holocentric chromosomes in angiosperms, this reliable exper-
imental method was first applied to Luzula (Juncaceae) by the
Portuguese cytogeneticists de Castro et al. (1949).
Besides X-rays (Roentgen rays), gamma rays can also be
applied to seeds, flower (inflorescence) buds, rhizome buds or
young root tips to study putative holocentric plants. Using this
method, the presence of chromosome fragments in subsequent
cell generations and the absence of potential micronuclei are
considered proof of holocentrism (La Cour 1953; Godward
1954; Håkansson 1954; Nordenski€ old 1963, 1964; Flach 1966;
Bokhari 1976; Tanaka and Tanaka 1979; Sheikh et al. 1995;
Vanzela and Colaço 2002). The presence of holocentric
chromosomes can be further confirmed by immunostaining
methods using a labelled antibody designed to bind one of the
proteins in the centromere/kinetochore complex (usually
CENH3). In the case of holocentric chromosomes, the signal
from the labelled antibody is present along the whole chromo-
some, whereas the signal is localized only at the centromere in
monocentric chromosomes (Nagaki et al. 2005; see
Sect. 12.4). Using light microscopy, the holocentric nature of
chromosomes can also be observed at mitotic metaphase,
when the absence of a primary constriction is more apparent,
and the sister chromatids are oriented parallel to the equatorial
plate (Fig. 12.1b). The positioning of the chromatids during
mitotic anaphase, when they are pulled broadside towards
opposite poles due to the attachment of the spindle fibres
along the entire length of the chromosome is also typical for
holocentric chromosomes (Fig. 12.1c). In contrast, during
P. Bureš et al.
anaphase of monocentric mitosis, a typical “V-shape” of
chromosomes can be observed due to the specific binding
of microtubules to the centromere. The parallel segregation
of holocentric chromosomes results in the formation of dupli-
cate anaphase plates, suggesting that the metaphase plate has
been split equatorially into two mirrored halves.
12.3 Occurrence of Holocentric
Chromosomes in Plants and Other
Organisms
Holocentrism is a stable, species-specific feature.2 Although
this phenomenon is much rarer than monocentrism, its occur-
rence is not randomly distributed, but phylogenetically clus-
tered in several eukaryotic lineages. It is a synapomorphic trait
for some families, genera, and subgenera in land plants and for
some classes, orders and families in animals.
In early diverging angiosperms, holocentrism has been
identified in Myristica fragrans (Myristicaceae,3 Magnoliales,
Flach 1966). In the monocot clade, it has been found in the sister
families of Juncaceae4 and Cyperaceae5 (Poales, de Castro
et al. 1949; La Cour 1953; Håkansson 1958; Nordenski€ old
2
A similar state, sometimes considered to be transitional between
monocentrism and holocentrism, is the formation of a neocentromere,
a chromosomal region that differs in its sequence and structure but can
function like a centromere in meiotic chromosomes. However, the
neocentromere in plants is more likely just an aberrant structure that
occurs occasionally in some individuals or populations (Dawe and Hiatt
2004). The majority of plant neocentromeres are the outcome of mei-
otic drive (Dawe and Hiatt 2004), which is one of the possible
mechanisms that has been suggested to explain the origin of holocentric
chromosomes (see Sect. 12.8).
3
Holocentric chromosomes have been studied in detail only in the
economically important nutmeg tree Myristica fragrans. Although
chromosome counts have been reported for seven additional species
in Myristicaceae, accounting for approximately 2% of the species
richness of the family (Bolkhovskikh et al. 1969; Goldblatt and Johnson
2010), the lack of detailed cytological analysis means that the presence
of holocentric chromosomes is unclear.
4
Although holocentrism has been experimentally confirmed several
times in the genus Luzula, it has been proposed to exist but not
satisfactorily proven in the genus Juncus (Godward 1985) and other
monotypic genera of Juncaceae (but see Sect. 12.7.3). In addition, in the
closely related family Thurniaceae (comprising four species of Thurnia
and Prionium), there is currently no karyological research so it is
unknown whether holocentric chromosomes are present here
(Bolkhovskikh et al. 1969; Goldblatt and Johnson 2010).
5
This represents the third most species-rich monocot family after
orchids and grasses. Numerous studies from the latter half of the
twentieth century reported localized centromeres in Cyperaceae from
S Asia, but these studies were rightfully doubted by Greilhuber (1995),
who considered them to be based on chromosomal constrictions
observed during mitotic prophase or metaphase, when chromosomes
are not entirely contracted. Holocentrism has since been definitively
confirmed experimentally in several taxa and should thus be considered
as a family synapomorphy (Greilhuber 1995).
12 Holocentric Chromosomes
1963, 1964) and separately in the genus Chionographis
(Melanthiaceae,6 Liliales,7 Tanaka and Tanaka 1979). In
the eudicot clade of angiosperms, it is present in the genera
Cuscuta8 (Convolvulaceae, Solanales, Pazy and Plitmann
1994) and Drosera (Droseraceae,9 Caryophyllales, Sheikh
et al. 1995). In total, holocentric chromosomes are estimated
to occur in almost 5,500 angiosperm species (approxi-
mately 1.5–2% of flowering plants), covering a wide
variety of life strategies including trees (Myristica),
perennial graminoids or annual herbs (Juncaceae,
Cyperaceae), parasites (Cuscuta), bulbous geophytes
(Chionographis), and carnivorous hemicryptophytes
(Drosera). Holocentric plants exhibit variation in breeding
strategies because although many are hermaphrodites, they
also include some dioecious taxa (Myristica, some species
of Carex, Kobresia, Didymiandrum and Scirpus) and
even gynodioecious taxa (Chionographis, Eriophorum
vaginatum, Juncus kraussii, and J. roemerianus). Agamo-
spermy alone has never been satisfactorily confirmed
(when tested) in holocentric plants. There is no apparent
geographical preference among holocentric taxa. Although
the most species-rich holocentric genus Carex, which
accounts for almost 2,000 species, is distributed primarily
in the temperate to the arctic climatic zones of the Northern
hemisphere, many other holocentric genera occur prefer-
entially in tropical areas.
Outside the angiosperms, holocentric chromosomes have
been reported in some green algae (Desmidiales and
Zygnematales; Godward 1954; King 1960) but so far they
6
The subfamily Chionographioideae comprises two monotypic
genera—Chionographis and Chamaelirium. Given the presence of
holocentric chromosomes in Chionographis, it has been suggested
that Chamaelirium luteum may also possess such chromosomes. How-
ever, cytological studies have so far failed to show whether the 18
chromosomes, which are very small compared with other species in
Melanthiaceae, are holocentric or monocentric (Thomas Meagher, per-
sonal communication).
7 Vanzela and Guerra 2000; Cuscuta: Guerra and Garcı́a
The putative holocentrism reported by Chakravorti (1948a, b) in the
families Zingiberaceae and Musaceae from the monocots was not
confirmed in later studies.
8
Holocentrism is present in the Cuscuta subgenus Cuscuta, whereas
monocentrism has been confirmed in the subgenus Monogyna.
Recently, holocentric chromosomes were also found in some taxa of
the subgenus Grammica (Guerra et al. 2010), in which monocentrism
was expected. In the subgenus Grammica, the sizes of the genomes and
chromosomes are highly divergent (McNeal et al. 2007), which could
indirectly suggest the presence of holocentrism (see Sect. 12.7.1).
9
In Aldrovanda, a related monotypic genus of Droseraceae,
chromosomes are very small and isodiametric without any apparent
primary constriction, but their holocentric nature has never been satis-
factorily confirmed. The monotypic genus Dionaea from the same
family, however, possesses monocentric chromosomes, as does
Drosophyllum, a monotypic genus recently separated into a different
monotypic family.
189
have never been found in gymnosperms, ferns or
bryophytes.10 Among animals, holocentric chromosomes
are widespread throughout several invertebrate groups,
including Nematoda, Onychophora, Dermaptera, Heter-
optera, Sternorrhyncha, Auchenorrhyncha, Lepidoptera,
Odonata, Phthiraptera, Psocoptera, Trichoptera, and Zorap-
tera, and are occasionally found in some families or orders in
Arachnida or Chilopoda. So far they have not been reported
in vertebrates (reviewed in more detail by Mola and
Papeschi 2006).
Because the majority of karyological studies in plants
have been limited to counting chromosomes, monocentrism
seems to be the predominant state among angiosperms. In
part this is because it has automatically been assumed to be
present in all taxa in which holocentric chromosomes have
not been proven using other cytogenetic techniques. In fact,
in addition to the group of taxa in which holocentrism has
been conclusively identified, it is possible that it may also be
present within genera which have been assumed to be all
monocentric but not studied in detail. For instance, in the
largely monocentric genus Cuscuta, it was only through
detailed karyotype analysis that holocentric chromosomes
were identified in subgenus Cuscuta.
12.4 Chromatin Structure of Holocentric
Chromosomes
An initial study by Ray and Venketeswaran (1978) analysed
the distribution of constitutive heterochromatin and deter-
mined that C-bands were dispersed along the entire length of
the holocentric chromosomes of Luzula elegans (¼
L. purpurea) and L. multiflora, in contrast with the centro-
meric location of C-bands in Vicia faba, the representative
monocentric organism used by these authors. However, the
dispersed pattern of heterochromatin was not confirmed in
subsequent investigations of holocentric taxa using C- or Q-
banding (Drosera: Sheikh and Kondo 1995; Rhynchospora:
2004). In the chromosomes of these holocentric taxa, hetero-
chromatin was observed to be localized in the terminal or,
less frequently, interstitial or median regions. This is there-
fore not so different from species with monocentric
chromosomes where, in addition to the centromeric location
that prevails in small chromosomes (<3 mm), terminal or
interstitial heterochromatin regions are also frequently
observed (Guerra 2000).
10
The unclear centromeric status reported by Vaarama (1954) in the
moss Pleurozium schreberi which is sometimes considered to be
holocentric, is actually neocentromeric activity (Dawe and Hiatt 2004).
190
No clear differences between holocentric and monocentric
chromosomes were found in either the location of ribosomal
DNA (rDNA) arrays or the portion of rDNA arrays associated
with the nucleolar organizer (Greilhuber 1995; Vanzela et al.
1998, 2003; Guerra and Garcı́a 2004; da Silva et al. 2010).
Nevertheless, substantial structural differences between
holocentric and monocentric plant chromosomes have been
recognized in (1) the amount and distribution of the kineto-
chore/centromeric proteins, (2) the number and location of
centromere-like satellites and (3) the phosphorylation pattern
of the canonical histone H3, which is temporally and spatially
associated with the cohesion of sister chromatids.
The proteins of the kinetochore/centromere complex are
conserved across all eukaryotes. The centromere-specific
variant of histone H3 (CENH3) plays a key role in this
complex. In monocentric chromosomes, CENH3 is
interspersed with canonical H3 only at the centromeres,
deliminating the boundaries of the centromere (Jiang et al.
2003). To see whether a similar pattern was also found in
species with holocentric chromosomes Nagaki et al. (2005)
studied the distribution of LnCENH3 (= CENH3 in Luzula
nivea). As expected, immunostaining with an antibody
against LnCENH3 confirmed previous ultramicroscopic
observations which showed that the functional kinetochore
extends along the entire length of the poleward faces of the
mitotic metaphase chromosomes, with the exception of the
telomeric regions (Bokhari and Godward 1980). In cross-
section, in which the chromosome is viewed axially (end-
on from the telomere), the kinetochore was observed to be
embedded in the outside groove, that runs along the chromo-
somal axis (Bokhari and Godward 1980; Nagaki et al. 2005).
This groove-embedding of the kinetochore seems to be com-
mon, at least in the genus Luzula, given that it was also
observed in the transverse sections of mitotic chromosomes
in Luzula elegans (¼ L. purpurea; Braselton 1971).
Unlike monocentric organisms, in which the amount of
CENH3 is constant throughout the cell cycle, the amount of
LnCENH3 varies throughout the cell cycle in Luzula nivea.
During interphase, LnCENH3 is clustered in small, dis-
persed areas, and its levels increase significantly from pro-
phase to metaphase and then decrease from anaphase to
interphase (Nagaki et al. 2005). A similar pattern has also
been observed in the holocentric nematode model organism
Caenorhabditis elegans (Buchwitz et al. 1999), suggesting
that varying amounts of CENH3 during the cell cycle may
provide a way to distinguish holocentric chromosomes from
monocentric ones.
In contrast to the conservation of centromere/kinetochore
proteins across eukaryotes, centromeric DNA is highly vari-
able in (1) the arrangement and number of satellite repeats and
(2) the length and nucleotide sequence of the respective
monomers. The amount of centromeric DNA also varies
widely between and within species, from tens of kilobases to
P. Bureš et al.
several megabases. Large arrays of centromeric satellite
repeats may be intermingled with centromeric retrotransposons
(Jiang et al. 2003; Ma et al. 2007), as observed in grasses with
specialized centromeric retrotransposons of the Ty3-gypsy
superfamily. In monocentric chromosomes, the length and
motif of the centromeric satellite DNA monomer may be
species-specific. Examples of this species specificity are the
180 bp pAL1 repeat in Arabidopsis thaliana (Nagaki et al.
2003), the 156 bp CentC repeat in maize (Ananiev et al.
1998) and the 155 bp CentO repeat in rice (Cheng et al. 2002).
Centromeric satellites have also been found in
holocentric chromosomes. Based on the homology to
RCS2 (rice centromeric sequence 2, another name for
CentO), Haizel et al. (2005) isolated and characterized a
Luzula centromeric satellite (LCS1) from Luzula nivea
(Juncaceae). In this species and nine other congeners, the
LCS1 monomer was shown to range in length from 173 to
178 bp and was arranged in tandem arrays within the
genome. The total size of these tandem arrays was about
50 kb, which is less than the shortest known centromeric
satellite array in flowering plants (65 kb in centromere 8 of
Oryza sativa: Cheng et al. 2002). It is possible that the
uncommonly short LCS1 arrays may be connected with the
origin of holocentric chromosomes (see Sect. 12.8).
LCS1 arrays in chromosomes of Luzula nivea are unique
not only in size, but also in location. They occur along all 12
chromosomes of L. nivea, with at least five clusters per
chromosome, whereas the centromeric satellite arrays of
monocentric organisms are restricted to the core of the
localized centromere. The arrays of LCS1 are associated
with heterochromatin and co-localize with the kinetochore,
suggesting that they are incorporated into regions that func-
tion as centromeres in holocentric chromosomes (Haizel
et al. 2005). During interphase, the centromeric histone
homolog LnCENH3 may be associated with LCS1, and
together, this complex may determine the chromosomal
regions from which the formation of the linear-shaped kinet-
ochore begins. However, the assembly of the kinetochore
along the whole chromosome raises a question as to how
the formation of the kinetochore can span euchromatic,
expressed regions. In Caenorhabditis elegans, this problem
seems to be solved by a mechanism related to RNA interfer-
ence. Initially, the transcripts from protein-coding regions
become substrates for RNA-dependent RNA polymerase,
which creates new antisense transcripts. Subsequently, ribo-
nuclease cleaves the newly formed transcripts to generate
small RNAs that are antisense to the original transcripts of
the protein-coding genes. These small RNAs interact with
specific proteins, and together, they stabilize chromatin and
kinetochores, similar to the pericentromeres of monocentric
chromosomes (Claycomb et al. 2009). It is likely that a
similar mechanism based on the RNA interference-related
pathway also works in holocentric plants.
12 Holocentric Chromosomes
The cell cycle-dependent phosphorylation of canonical
histone H3, which seems to be required for the cohesion of
sister chromatids (Gernand et al. 2003; Houben et al. 2007),
is another feature that distinguishes the structure of
holocentric and monocentric chromosomes. In monocentric
plants, the phosphorylation of H3 is restricted to the
pericentromeric regions during mitosis and meiosis II,
whereas H3 is phosphorylated along the entire chromosome
during prophase I, when the sister chromatids are held
together along their whole length (Manzanero et al. 2000).
In contrast, in holocentric plants such as Luzula nivea
and L. luzuloides (Juncaceae) or Rhynchospora tenuis
(Cyperaceae), the phosphorylation of H3 occurs along entire
chromosomes during both mitosis and meiosis (Gernand
et al. 2003; Nagaki et al. 2005; Guerra et al. 2006).
12.5 Mitosis in Holocentrics
Upon entry into mitosis, both monocentric and holocentric
chromosomes become more compact in structure (reviewed
by Stack and Anderson 2001), and sister chromatids must be
held together to prevent aneuploidy caused by premature
segregation. In this process, condensin and cohesin proteins
have a key role. Condensin I facilitates the general conden-
sation of chromatin, while condensin II creates a rigid chro-
mosome structure (Maddox et al. 2004). The adhesion of
chromatids is ensured by cohesins (Ono et al. 2003).
The stronger tension of holocentric chromatids due to the
large target area for microtubule attachment creates a special
requirement for more compact condensation and more
extensive cohesion compared with monocentric chromatids.
The more rigid structure of holocentric chromatids is
ensured by the dominant activity of the condensin II com-
plex along the entire length of the chromatid. In monocentric
chromosomes, condensin II is enriched only in the centro-
mere/kinetochore region, so their arms are not as rigid as
those in holocentric chromosomes. Extensive cohesion
along the whole length of sister chromatids ensures their
close parallel orientation before spindle attachment and
that their distance does not dramatically increase when bipo-
lar attachments form (Maddox et al. 2004). Theoretically,
the large surface area of the kinetochore in holocentric
chromosomes increases the likelihood of attachments to
the microtubules emanating from the opposite spindle
poles (merotelic attachment), which could result in improper
chromosome segregation and, thus, aneuploidy (Knowlton
et al. 2006). However, the lengthwise cohesion of rigid
chromatids leads to a fixed (back-to-back), bipolar chroma-
tid orientation and thus restricts the attachment of a particu-
lar kinetochore to a single spindle pole, ensuring that each of
the sister kinetochores attaches to the opposite spindle pole
(Stear and Roth 2002).
191
The structure of the diffuse kinetochore is very similar to
the organization of the classical trilaminar kinetochore
identified in monocentric chromosomes using electron
microscopy (Rieder 1982). The gradual formation of the
kinetochore can be visualized using the centromere/kineto-
chore protein CENH3. While CENH3 occupies discrete
domains during interphase (Fig. 12.1a), these regions move
closer to form a continuous lengthwise kinetochore at the
poleward side of each chromatid during mitosis (Fig. 12.1b).
Holocentric and monocentric chromosome behaviour is
roughly similar during prometaphase, in which kinetochores
attach end-on to dynamic microtubules to congress
chromosomes to a very tight metaphase plate. During meta-
phase, the holocentric sister chromatids are oriented parallel
to the equatorial plate, just as monocentric chromatids are.
During anaphase (Fig. 12.1c), sister chromatid cohesion is
resolved, and chromatids are pulled along their entire length
to the spindle poles. As a result, their parallel orientation
persists during the anaphase movement, and the holocentrics
lack the “V-shape” typical of monocentrics. Because the
sister chromatids are pulled towards the poles by the spindle
microtubules which act along the entire length of
the chromosomes neither the preferential orientation nor
the spatial localization of the telomeres (Rabl-orientation)
form in interphase nuclei.
12.6 Meiosis in Holocentrics
The main scenario for meiotic prophase I is the same in
holocentrics as in monocentrics. In both, sister11 chromatids
are interconnected over their whole length by cohesion and
homologous chromosomes align to enable the formation of
the synaptonemal complex and recombination via crossover.
11
In mitosis, the sister chromatids are clearly defined both in
holocentrics and monocentrics as two identical (daughter) chromatids
derived from duplication (replication) of one and the same chromosome
during of the cell cycle [see e.g., King RC, Stansfield WD, Mulligan PK
(2006) A dictionary of genetics. 7th Ed. Oxford Univ. Press, Oxford].
In meiosis, the term “sister” regarding to patchwork chromatids
recombined via crossover is not so easy to define in holocentrics as in
monocentrics where connection of centromeres defines which two
chromatids are sister while those which are not joined by a common
centromere are non-sister (¼ homolog) chromatids [see e.g., Brooker
RJ (2009) Genetics: analysis & principles. 3rd Ed. McGraw-Hill Irwin,
New York, p. 49, 821]. In holocentrics, the “majority rule” defines
sister and non-sister chromatids meaning that sister chromatids share
longer identical segments originated via replication, while non-sister
chromatids share shorter absolutely identical segments. From a practi-
cal viewpoint, the terms “sister” and “non-sister” well fitted to mitotic
chromosomes are universally conserved by such additional criteria
(common centromere or “majority rule”) for any type (mitosis or
meiosis, holocentric or monocentric) or phase (pre- or postrecombi-
national) of cell division to overcome the difficulty that no original
sister chromatids are present anymore after recombination.
192
Each round of meiotic division in both chromosomal types
is associated with one-step loss of cohesion between
chromatids. The main differences between holocentric and
monocentric chromosomes are (1) the way in which sister
chromatid cohesion is lost, (2) the number of crossover sites
per bivalent and (3) the orientation of the chiasma within the
bivalent to manage the segregation.
In monocentrics, sister chromatid cohesion in the arms is
released, and only the centromeric regions remain connected
during meiosis I. The connection between non-sister
chromatids is retained at the chiasmata while the remaining
internal cohesion of the sister chromatids is protected at the
centromeres by members of the MEI-S332/Shugoshin protein
family (Sakuno et al. 2009). Moreover, the sister kinetochores
are covered by the kinetochore protein MIS12, which causes
both kinetochores to present a shared face to the microtubule-
binding proteins and allows the “co-orientation” of the sister
chromatids during meiosis I (Li and Dawe 2009). This “co-
orientation” is later changed to a “bi-orientation”, and the
remaining cohesion between the centromeres is resolved dur-
ing meiosis II, similar to mitotic division.
Holocentric chromosomes lack a predefined site for the
protection of cohesion in meiosis I, so the chiasma serves as
an alternative point of reference. In contrast to monocentrics,
the number of crossover sites appears to be limited in
holocentrics, occurring only once or twice per bivalent
(White 1973). Indeed, a deleterious effect caused by the
formation of more than two chiasmata was observed by
Nokkala et al. (2004). Each chiasma persists to the end of
metaphase I, holding the four chromatids in a cross-shaped
bivalent (Fig. 12.2b), and following further condensation
this gives rise to the so-called “box configuration”
(Fig. 12.2c, d, g; Nordenski€ old 1962). The orientation of
the meiotic bivalents on the metaphase plate mediates the
stepwise loss of sister chromatid cohesion. The cohesion is
released in those parts of the bivalent lying on the equatorial
plate (shown in yellow in Fig. 12.2d, g), allowing segrega-
tion toward the opposite poles during meiosis I, whereas
those chromatid parts lying above and below the equatorial
plate (oriented axially) remain connected (Fig. 12.2e, h).
Because there is an absence of detailed knowledge about
meiosis in plant holocentrics, the detailed studies in
Caenorhabditis elegans can serve as a model to show how
the crossover position can direct the holocentric chromo-
some orientation. It should be noted that C. elegans is an
animal model and that molecular mechanisms described
below might differ from those of holocentric plants, espe-
cially in the proteins involved in meiotic division, although
it is likely that general principles would be similar. More-
over, C. elegans exhibits telokinetic meiosis whereas most
holocentric plants show holokinetic behaviour (see below
and Sects. 12.6.1 and 12.6.2). During meiosis in C. elegans,
the number of crossover sites is limited to a single chiasma
P. Bureš et al.
for each pair of homologs (Meneely et al. 2002). During late
prophase, the homologs desynapse and are remodeled into a
cross-shaped bivalent (Fig. 12.2a, b). The position of the
chiasma determines the shape of the bivalent where the longer
axis separates sister chromatids while the shorter axis
separates nonsister chromatids regarding majority rule (see
footnote 11). Cohesins and the HORMA proteins (HTP-3
and HIM-3) remain homogeneously distributed, connecting
chromatids on both the long and short arms of the bivalent
(Nabeshima et al. 2005). In contrast, the distribution of other
proteins is associated with particular chromosomal axes and
the anaphase-specific release or retention of these other
proteins specifies the direction of the bisection of the bivalent.
The meiosis-specific proteins HTP-1 and HTP-2 (of the
HORMA domain family) and LAB-1 (a nematode-specific
protein) are depleted on the short axis and are retained on the
long axis, where cohesion is retained until meiosis II (de
Carvalho et al. 2008; Martinez-Perez et al. 2008). In contrast,
the SYP proteins associate with the short arms, (where cohe-
sion is lost during meiosis I) (Pasierbek et al. 2001; shown in
yellow in Fig. 12.2g and hypothetically expected and shown
also in yellow in postreductional type in Fig. 12.2b, c, d), and
occupy domains where HTP-1/2 and LAB-1 are depleted (de
Carvalho et al. 2008; Martinez-Perez et al. 2008). The persis-
tence of SYP-1/2 on the short axis correlates also with the
recruitment of the Aurora-like kinase AIR-2, which directs the
loss of sister chromatid cohesion through the phosphorylation
of the cohesin subunit REC-8 and its subsequent cleavage by
separase (Rogers et al. 2002). This spatiotemporal protein
diversification allows sister chromatids to switch between the
“co-orientation” and the “bi-orientation” during meiotic
anaphases.
Whereas the attachment of microtubules in mitosis
always occurs along the whole poleward face of each chro-
matid (holokinetic), two types of microtubule attachments
exist in meiosis. Depending on whether the orientation of the
meiotic bivalent is equatorial or axial, microtubule attach-
ment is holokinetic (Fig. 12.2e, f) or telokinetic (Fig. 12.2h, i),
respectively.12 Both types differ from conventional mono-
centric meiosis, which is pre-reductional (i.e the reduc-
tional division is followed by the equational division)
(Fig. 12.2j–l). Assuming two alternative successions of the
12
As a consequence of the fission or fusion of chromosomes, trivalents
are sometimes formed during meiosis: two fragments are homologous
to one non-fragmented chromosome, or one fused chromosome is
homologous to the two non-fused ones. Two types of segregation are
possible in this case. In the first case (telokinetic meiosis), the larger
chromosome migrates to one pole, whereas the two shorter
chromosomes migrate to the other pole during meiosis I. In the second
case (holokinetic meiosis), two triads of individual chromatids segre-
gate. This specific situation, if observed, could be also used to identify
the holokinetic or telokinetic type of meiosis.
12 Holocentric Chromosomes
Fig. 12.2 Comparison of meiosis in holocentric (a–i) and monocentric
(j–l) organisms. Sister segments of chromatids are represented by the
same color, either grey or blue, kinetochores and microtubules are
black, cohesion between chromatids is indicated in red, proteins asso-
ciate specifically with the axis laying in the equatorial plate are in
yellow (in d [Luzula] hypothetically expected based on analogy with
Caenorhabditis), and the equatorial plate is indicated by the dotted line.
Holocentric chromosome behaviour is shown in prophase I (a–c; here
postreductional type), holokinetic meiosis (d–f; Luzula), telokinetic
meiosis (g–i; Caenorhabditis), monocentric meiosis (j–l), metaphase
I (d, g, j), anaphase I (e, h, k), and anaphase II (f, i, l). (a) Removal of
the synaptonemal complex and the remodelling of chromosomes. (b)
Cross-shaped bivalent. (c) The “box configuration” of the bivalent
resulting from the further condensation of the chromosome and the
divisions, the post-reductional type was identified in
holocentric species, in contrast to the pre-reductional in
monocentrics (Wahl 1940). Actually, regarding the position
of the crossover site in the telokinetic (pre-reductional)
meiosis, the cohesion proteins are released from the short
193
chiasma resolution. (d) Equatorial orientation of bivalents. (e) “Sister”
chromatids (corresponding to larger sister segments) segregate (equa-
tional step), while half-bivalents (short axis) remain connected. (f)
Chromatids are dragged broadside (reductional step). (g) Axial orien-
tation of a bivalent. (h) “Sister” chromatids (corresponding to larger
sister segments) move together (reductional step) pulled by the chro-
mosome ends. (i) Chromatids segregate, pulled by their ends, which
were inactive during meiosis I (equational division). (j) Equatorial
orientation of the bivalent; sister parts of chromatids are connected.
(k) Chromosome homologs segregate, retaining the junction only in the
centromeres (reductional step). (l) Sister chromatids segregate, pulled
at the centromeres (equational division)
axis so that the whole homologous chromosomes move to
opposite poles. In contrast, holokinetic meiosis begins with
the separation of the long axis (the sister chromatids), mean-
ing that the reductional step occurs during the second divi-
sion. Thus, the terms holokinetic and telokinetic seem to be
194
more appropriate for the description of chromosomal mei-
otic movement in holocentrics. Regardless of the type of
chromosomal movement (holokinetic, telokinetic, or
monokinetic), the final product of meiosis (i.e., four
gametes), is the same.
12.6.1 Holokinetic Meiosis
Most data on holokinetic meiosis have been obtained from
Juncaceae (Nordenski€ old 1962), Cyperaceae (Da Silva et al.
2005) and Cuscuta (Convolvulaceae; Pazy and Plitmann 1987,
1994). Although this type of meiotic behaviour seems to be
typical for plants with holocentric chromosomes, it has also
been observed in some animals, for example, during meiosis in
the female mealybug Planococcus citri (Bongiorni et al. 2004).
The segregation of sister chromatids has also been documented
for univalent chromosomes, such as the X and Y chromosomes
of some holocentric insects (Nokkala et al. 2004) and the B
chromosome of Cuscuta babylonica (Pazy 1997).
During metaphase I, the long axes of the cross-shaped
meiotic bivalents lie in the equatorial plate so that most of
the sister chromatid cohesin is released upon anaphase I
(Fig. 12.2d). This type of release allows the “bi-orientation”
of sister chromatids and their segregation to opposite spindle
poles (equational division; Fig. 12.2e). Connections along
the non-sister chromatids (short axis) are retained at the end
of the chromosome in a small region connected with chi-
asma (shown in red in Fig. 12.2e). This chromosome struc-
ture is also known as a half-bivalent or demi-bivalent (Pazy
and Plitmann 1987). The kinetochore plate covers the entire
chromatid in a manner similar to that seen during the
holocentric mitotic anaphase and the chromatids are
pulled along their entire length (Nordenski€ old 1962, 1963).
In Luzula species, homologous chromatids re-associate
during the short meiotic interphase and remain paired
until the end of metaphase II (Nordenski€ old 1962), enabling
them to become correctly oriented to opposite poles
during metaphase II and to subsequently segregate in a
holokinetic manner during anaphase II (Fig. 12.2f).
In contrast, only telomeric associations between homolo-
gous chromatids remain until the second meiosis in Cuscuta,
enabling a reductional division (Pazy and Plitmann
2002). Both of these types of chromosome associations
(i.e., re-associated and only terminally associated) were
observed by Strandhede (1965a) in Eleocharis palustris
subsp. palustris.
12.6.2 Telokinetic Meiosis
The appropriate model of telokinetic meiosis is the
nematode Caenorhabditis elegans (Maddox et al. 2004;
P. Bureš et al.
Schwarzstein et al. 2010). In C. elegans, as noted above,
only one crossover forms between homologs (Meneely et al.
2002), and the cross-shaped meiotic bivalent is oriented
axially so that the short axis lies in the equatorial plate
(Fig. 12.2g; Albertson and Thomson 1993). In telokinetic
meiosis, the kinetic activity of the holocentric chromosomes
changes from dispersed in mitosis to localized in meiosis,
and the kinetochore forms a cup-like shape (Dumont et al.
2010). This conformation ensures the encasement of sister
chromatids together to promote their movement as a single
functional unit during meiosis I (Monen et al. 2005), similar
to the connection of the monocentric sister kinetochores (Li
and Dawe 2009). The ends of the chromosomal axes repre-
sent two potential “localized meiotic centromeres” that can
mediate chromosome spindle interactions. Depending on the
crossover site, one end becomes active. Thus, the active
chromosome end is oriented to the spindle pole during
meiosis I (Fig. 12.2h: end A), and the other end (Fig. 12.2i:
end B) is then active during meiosis II.
In C. elegans, the kinetic activity is localized at the
chromosome end that is opposite to the chiasma, whereas
in the insect Triatoma infestans (Heteroptera), the kinetic
activity may alternatate between the chromosome ends,
which are differentiated by the presence or absence of a
terminal C-band, irrespective of the chiasma position
(Pérez et al. 1997). Similarly, in different somatic cell
types of the nematode Parascaris univalens, the kinetic
activity can be associated with either heterochromatin or
euchromatin (Goday and Pimpinelli 1989), indicating that
the kinetic activity in holocentric chromosomes is not depen-
dent on a specific nucleotide sequence but rather is epige-
netically controlled (Sullivan et al. 2001). A clear example
of a telokinetic plant species is from Chionographis
(Melanthiaceae).13 Tanaka and Tanaka (1979) determined
that there were four Japanese Chionographis taxa with
holocentric chromosomes. The meiotic behaviour of two of
these—C. japonica and C. japonica var. hisauchiana—was
later (Tanaka and Tanaka 1980) studied, revealing that
Chionographis bivalents orient axially during meiosis and
undergo reductional separation in the first meiotic division
and equational division in the second.
13
In plants, telokinetic meiosis was reported in Luzula elegans by
Malheiros et al. (1947), who suggested that during anaphase I and II,
the kinetic activity is restricted to both ends of each chromosome. This
species was later analysed more precisely by Östergren (1949), who
claimed that L. elegans chromosomes undergo inverted meiosis (i.e.,
holokinetic behaviour). The holokinetic chromosomal behaviour has
also been reported in other species of Luzula (Nordenski€old 1962,
1963; Braselton 1971, 1981).
12 Holocentric Chromosomes
12.7 Holocentric Karyotypes and Their
Evolution
The range of chromosome numbers encountered in
holocentric plants is almost as wide as that in monocentric
plants. Despite the rarity of holocentrism (see Sect. 12.3), we
find that within the very small group of six angiosperm
species which possess the lowest chromosome number so
far reported in angiosperms (n ¼ 2),14 the holocentric spe-
cies Rhynchospora tenuis (Vanzela et al. 1996). Indeed, very
low chromosome numbers are not rare among other
holocentric species, as n ¼ 3 has been recorded in
Eleocharis subarticulata, Fimbristylis narayanii, F.
umbellaris, Luzula elegans, and Drosera roseana
(Malheiros-Garde and de Castro 1947; Rath and Patnaik
1978; Nijalingappa and Tejavathi 1984; Kondo et al. 1994;
da Silva et al. 2005), and n ¼ 4 has been found in Cuscuta
babylonica (Pazy and Plitmann 1987, 1994). At the other
end of the scale, we find some holocentric plants with
extremely high chromosome numbers, such as some taxa
of the family Cyperaceae including Eleocharis kuroguwai
(n ¼ c. 98; Hoshino 1987), Cyperus pangorei (n ¼ 104;
Nijalingappa and Tejavathi 1984), and C. cyperoides
(n ¼ 110; Tejavathi 1988). Similarly, a wide range of chro-
mosome numbers seems to be common in holocentric
animals. For example, the extremely low chromosome num-
bers of n ¼ 1 and n ¼ 2 have been reported in the germline
cells of the nematode Parascaris univalens (Ascaridae;
Goday et al. 1988) and in the insect Lethocerus sp.
(Belostomatidae, Heteroptera; Papeschi and Bressa 2006),
respectively, whereas exceptionally high numbers of
chromosomes have been found in some butterfly species:
n ¼ 147150 in Plebicula dorylas (Lepidoptera,
Lycaenidae; Kandul et al. 2004), n ¼ c. 150 in Abananote
erinome (Lepidoptera, Ithomiidae; Brown et al. 2007), and
n ¼ 223 in Polyommatus atlantica (¼ Lysandra atlantica;
Lepidoptera, Lycaenidae; de Lesse 1970). The latter is the
highest chromosome number so far reported for any animal
(Marec et al. 2009). Holocentric desmid algae also exhibit
chromosome numbers that vary extensively, ranging from
n ¼ 2 in Spirogyra cylindrica to n ¼ c. 200 in
Micrasterias rotata, and n ¼ 592 in Netrium digitus (King
1960). As in monocentric plants, both polyploidy and chro-
mosomal rearrangements are likely to be the main forces that
increase the range of chromosomal variation. In animals,
however, where polyploidy is less frequent, chromosomal
14
Such a low chromosome number has been found in only five other
angiosperms: Zingeria biebersteiniana, Colpodium versicolor,
Brachycome dichromosomatica, Haplopappus gracilis, and
Ornithogalum tenuifolium (Cremonini 2005).
195
Table 12.1 Rate of chromosomal rearrangement in holocentric and
monocentric taxa
Chromosome type/taxon Rearrangement ratea
Holocentrics
Lepidoptera 1.370–3.300b
Nematoda 0.500–0.700b
Monocentrics
Diptera 0.05253–0.08485c
Brassicaceae 0.00357–0.01071c
Poaceae 0.00013–0.00041c
Solanaceae 0.00010–0.00029c
Mammalia 0.00006–0.00030c
a
Number of chromosomal breakages (Mb/millions of years); not
calibrated by generation time
b
Data taken from d’Alençon et al. (2010)
c
Data taken from Ranz et al. (2001)
rearrangements are likely to generate the wide range of kar-
yotype variation more effectively in holocentric taxa than in
monocentric ones. Indeed, a much higher chromosomal rear-
rangement rate was detected in holocentric organisms, such as
the nematodes and butterflies, compared with monocentric
organisms (Brassicaceae, Poaceae, Solanaceae, Mammalia;
Table 12.1) using the number of chromosomal disruptions in
the available genome sequences of the taxa in relation to their
genome size (Mb) and divergence time (millions of years).
Within groups of related monocentric plants, a pure arith-
metic relationship among chromosome numbers is usually
sufficient to detect recent polyploidy events (or true aneu-
ploidy15). However, in holocentric taxa, this empirical pattern
is often obscured by frequent chromosomal rearrangements,
particularly chromosomal fissions (agmatoploidy) and fusions
(symploidy). Moreover, in some holocentric organisms, the
relatively clear presence of polyploidy could be illusory due to
complete agmatoploidy (concerted fission), which occurs, for
example, in Luzula (see Sect. 12.7.3.). Therefore, chromosome
counting must be combined at least with the measurement of
DNA content to reliably evaluate the role of polyploidy in
holocentric taxa. A combination of both parameters, such as
average chromosome size (C/n ¼ 2C DNA/2n; Šmarda et al.
2008) could serve as a criterion in which stability (conditioned
by a linear relationship between 2C and 2n) indicates the
presence of polyploidy or true aneuploidy if chromosome
number is variable. Conversely, an increase or decrease in
C/n could suggest fusion or fission events, respectively (see
Nishikawa et al. 1984). Unfortunately, as has been observed in
some monocentric organisms, the proliferation or removal of
retrotransposons can also be very rapid over short evolutionary
scales in holocentric organisms, and can thus also be responsi-
ble for increases or decreases in chromosome size within
15
The gain or loss of a particular chromosome or chromosomes due to
non-disjunction during irregular meiotic chromosomal segregation.
196
genera, subgenera or other groups of related taxa independent
of chromosome number changes. The estimation of the
retrotransposon density in the genome of a particular species
is a further parameter that should be taken into consideration in
the study of holocentric karyotype evolution (see Zedek et al.
2010). Additionally, the study of multivalent formation during
the first meiotic metaphase or a comparative analysis of the
FISH patterns in particular species could be helpful for
detecting individual chromosomal fission/fusion events or
other chromosomal rearrangements, particularly at the intra-
specific level (see da Silva et al. 2005, 2008a).
12.7.1 Symploidy and Agmatoploidy
Although in monocentrics, chromosomal fission usually
results in the deletion of acentric fragments (see Lysák and
Schubert 2013, this volume), such usually deleterious effects
are largely prevented in holocentric species by the nature of
their chromosomes. Moreover, the extended kinetochore
reduces the risk of forming dicentric chromosomes, which
after fusion, are often lethal in monocentric organisms. Given
these observations and predictions it is perhaps not surprising
that agamatoploidy and symploidy can play prominent roles in
karyotype evolution of holocentric species compared with
monocentric ones. (Heilborn 1924; Luceño and Castroviejo
1991; Hipp 2007; Hipp et al. 2007; Escudero et al. 2008; Hipp
et al. 2009). In agreement with the higher rearrangement rate
detected in holocentric organisms (Table 12.1), the probabilis-
tic models of karyotype evolution in Carex (Cyperaceae) favor
the gain or loss of an individual chromosome as the most
probable mode of karyotype evolution in sedges, suggesting
that fission or fusion has a more important role than polyploidy
in this process (Hipp et al. 2009; Mayrose et al. 2010).
Recurrent chromosomal fission and/or fusion during kar-
yotype evolution can lead not only to a wide range of
chromosome numbers (see Sect. 12.7), but also to an almost
continuous (dysploid) series of chromosome numbers within
particular holocentric families, genera, sections or other
infrageneric taxa.16 Examples of karyotype variation in
some holocentric plant genera are given in Table 12.2. The
extremely broad and continuous karyotype variation on a
small evolutionary scales can also be found in animals with
holocentric chromosomes, such as the butterflies of
Nymphalidae (the most specious family of Lepidoptera)
where the chromosome number ranges nearly continuously
16
In some holocentric taxa, the wide range of intraspecific variation
may also be associated with true aneuploidy, for example, in Eleocharis
uniglumis s. l. (2n ¼ 4451, 5356, 5989, 92; Strandhede 1965b,
1966; Bureš 1998), where the chromosome number and DNA content
are linearly correlated (Bureš and Kneřová unpubl.). Other examples of
true aneuploidy in Carex are reviewed by Hipp et al. (2009).
P. Bureš et al.
from n ¼ 5 to n ¼ 134 (Brown et al. 2004, 2007; Marec
et al. 2009). Furthermore, out of all the animal genera which
have been examined cytologically, the 48-fold range in
chromosome numbers in the insect genus Apiomorpha
(n ¼ 296; Hemiptera, Eriococcidae; Cook 2000) and the
13.4-fold range encountered in the butterfly genus
Agrodiaetus (n ¼ 10134; Lepidoptera, Lycaenidae;
Lukhtanov and Dantchenko 2002; Lukhtanov et al. 2006)
are considered to be the most extensive (Cook 2000) and
both these genera possess holocentric chromosomes.
Indirect evidence that agmatoploidy and symploidy are
ongoing processes in plant species with holocentric
chromosomes is suggested by the occurrence of univalents
or heteromorphic trivalents, which are frequently observed
in meiosis I in several taxa of Cyperaceae, particularly Carex
(Wahl 1940; Tanaka 1949; Faulkner 1972; Hoshino 1981;
Luceño and Castroviejo 1991; Luceño 1994; Hoshino and
Waterway 1994; Escudero et al. 2008) and Eleocharis
palustris subsp. palustris (Strandhede 1965a). Furthermore,
intraplant variations in chromosome number suggest a high
frequency of fission or fusion, such as observed in Carex
disticha (Luceño 1992), Eleocharis palustris subsp.
waltersii (Strandhede 1965b, 1966; Bureš et al. 2004) and
in Luzula multiflora, L. nivea and L. luzuloides (Kuta et al.
2004), where the stability of DNA content contrasts strongly
with the extensive variability in chromosome number.
A detailed analysis of particular chromosomal fusions in
local populations of Eleocharis maculosa was performed by
da Silva et al. (2008a). These authors found three cytotypes
with 2n ¼ 6, 7, and 8, compared with the common cytotype
of 2n ¼ 10, and explained their origins by fusions of partic-
ular non-homologous chromosomes using a sophisticated
combination of conventional mitotic methods, FISH (using
45S rDNA, 5S rDNA and telomere probes) and an analysis
of meiotic chromosome pairing. The structural heterozygos-
ity of all three Eleocharis maculosa cytotypes suggests that
they could not be assumed to be stabilized, creating chromo-
somal races (see da Silva et al. 2008a). Although meiosis
could be irregular in holocentric structural heterozygotes
(possessing an unbroken chromosome together with its
homolog fragments or a fused chromosome with its non-
fused homologs), the newly fused (or fragmented) karyotype
could easily be homologized via selfing or backcrossing and
subsequently fixed, for example, by mechanisms of genetic
drift resulting in local chromosomal races. Indeed, this out-
come was observed in Carex conica by Hoshino and Water-
way (1994), C. sociata by Ohkawa et al. (2000), and
C. scoparia var. tessellata by Hipp et al. (2010).17 Some
17
The presence of more than one cytotype in the species was
documented in more than 100 species of Carex and many other taxa
of Cyperaceae (Roalson 2008; Hipp et al. 2009).
12 Holocentric Chromosomes 197

Table 12.2 Variation in chromosome number in some holocentric plant genera

Family/genus Range Haploid chromosome number (n)
Droseraceae
Droseraa, b, c 13.3-fold 3–7, 9–17, 20, 23, 30, 32, 36, 40
Melanthiaceae
Chionographisd 1.8-fold 12, 21, 22
Juncaceae
Luzulaa, b, e 14.0-fold 3, 6–16, 18, 21, 23, 24, 26, 31, 33, 35, 36, 42
Juncusa, b 10.6-fold 9, 10, 13, 15–24, 30, 32, 34, 35, 40, 42, 45, 50, 53, 54, 60, 66, 67, 85
Cyperaceae
Carexf, g 11.6-fold 5–47, 50, 52–58
Eleocharisf, h 36.0-fold 3–16, 18–30, 36–44, 47–50, 68, 86–92, 100, 108
Cyperusf 22.4-fold 5, 8, 9, 12, 13, 15–32, 34–45, 47–50, 52–60, 62–64, 66–69, 76, 80, 93, 98, 104, 110, 112
Rhynchosporaf, i 12.5-fold 2, 4–13, 15, 18–22, 24, 25
a
Bolkhovskikh et al. (1969)
b
Goldblatt and Johnson (2010)
c
Rivadavia et al. (2003)
d
Tanaka and Tanaka (1979)
e
Nordenski€old (1951); Kirschner (1992)
f
Roalson (2008)
g
Rotreklová et al. (2011)
h
Strandhede (1965b, 1966); Bureš (1998)
i
Luceño et al. (1998); Vanzela et al. (2000, 2003)

stable, species-specific bimodal karyotypes, such as those in
Eleocharis palustris subsp. palustris and E. mamillata (2n ¼
12M þ 4L; Strandhede 1965b; Bureš et al. 2004),
Fimbristylis complanata (2n ¼ 12M þ 4L; Yano and
Hoshino 2006), or Luzula alpina (2n ¼ 12AL þ 24BL;
Kirschner 1992; Bačič et al. 2007), could be assumed to
have originated via symploidy or agmatoploidy then fixed
through this mechanism. On the other hand, in holocentric
organisms, the degree of complexity of such karyotype
rearrangements should probably be higher than that in
monocentric organisms in order to create a sufficiently effec-
tive reproductive barrier necessary for speciation. This point
was argued by Hipp et al. (2010), who analysed the rate of
gene flow among populations within the sedge species Carex
scoparia and C. pachystachya, which both display extreme
inter-population divergence in chromosome number.
A rather intriguing question is whether these symploid or
agmatoploid “small steps” in the evolution of holocentric
karyotypes have resulted from a stochastic or a deterministic
process. The geographic trend in karyotype orthoselection18
can be inferred from the chromosome variation in Carex
laevigata, in which 2n increases from 69 in the Pyrenees
Mountains, continues to increase along the northern and
western shores, and reaches 84 at the southern tip of the
Iberian Peninsula (Luceño and Castroviejo 1991; Escudero
et al. 2008). Hipp et al. (2009), who also reviewed other
18
Karyotypic orthoselection occurs when a particular type of chromo-
somal rearrangement is present recurrently in a phylogenetic lineage
(White 1973). In this case, the presence of fission or fusion is assumed
to have occurred.
geographic patterns in chromosome variation in Carex, con-
sidered these intraspecific chromosomal geographic clines in
sedges to most likely have resulted from the stochastic
effects of chromosome divergence associated with species
range expansion rather than by any environmentally directed
adaptive selection in chromosome number.
A phylogenetic trend of decreasing chromosome number
associated with an increase in chromosomal size has been
suggested for Eleocharis. In this genus species with the
largest chromosomes are found in the evolutionary youngest
subgenus (Eleocharis subgen. Eleocharis), while those spe-
cies with numerous and very small chromosomes are found
in the early diverging phylogenetic lineages comprising
subgenera Limnochloa and Zinserlingia (Yano et al. 2004;
da Silva et al. 2008b; Zedek et al. 2010). A similar pattern
might also be present in Schoenus (Bhatti et al. 2007). In
contrast, the Luzula species with the largest chromosomes,
L. elegans, occupies a position that is sister to all other
Luzulas based on the latest phylogenetic tree of this genus
(Záveská-Drábková and Vlček 2010). In Carex, an evolu-
tionary trend from lower to higher chromosome numbers
was assumed by the early studies of Heilborn (1924)
and in light of recent phylogenetic data the Heilborn hypo-
thesis seems to hold generally over the broad evolutionary
scale. Carex species with the lowest numbers and largest
chromosomes (C. siderosticta, C. pachygyna, and C. ciliato-
marginata) belong to section Siderosticae which forms a
clade sister to the rest of the genus (Waterway et al. 2009).
In this genus, however, the opposite trend was also found at a
finer scale by Hipp (2007), who tested various probabilistic
models on the North American species of Carex sect. Ovales
and demonstrated that the processes of increasing and
198 P. Bureš et al.

180

Ratio largest / smallest average chromosome size within particular genus

160

140

120
5 µm
100
Luzula elegans c Luzula pilosa c
80

60
–––––––––––

40

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20 –––––––––––
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0
Monocentrics a Holocentrics b

Median 25%-75% Non-outliers ––––– Outliers and Extremes

Fig. 12.3 Divergence in chromosome size within monocentric and chromosome size, the ratio between the largest and the smallest chro-
holocentric plant genera in which the chromosome number and the mosome was calculated. (b) Eight genera are represented using data for
amount of nuclear DNA are known for at least two species. The inner Drosera, Scirpus, Cyperus, Carex, and Juncus from the Plant DNA
box illustrates the 168-fold difference in the size of chromosomes in the C-values Database (Bennett and Leitch 2005) and data for Eleocharis
genus Luzula. (a) 2C and 2n data for the 333 genera taken from the Plant (Zedek et al. 2010), Cuscuta (subgenera Cuscuta and Grammica;
DNA C-values Database (Bennett and Leitch 2005). For each species in McNeal et al. 2007) and Luzula (Barlow and Nevin 1976). (c) Taken
which the somatic DNA amount and chromosome number are both from Nordenski€ old (1951); Luzula elegans: 2n ¼ 6, 2C ¼ 8.55 pg;
known, the average chromosome size was calculated. Subsequently, L. pilosa: 2n ¼ 66, 2C ¼ 0.55 pg (Barlow and Nevin 1976)
for all genera containing at least two species with a known average

decreasing chromosome numbers were non-random and were due to the extreme variations in chromosome number
present in various phylogenetic lineages. This observation (x-axis). Surprisingly, this was not found, instead a plot of
suggested that different kinds of orthoselection may have data for 64 holocentric species showed that the relationship
modulated rapid karyotype shifts during the early divergence was negative; the more chromosomes, the smaller the genome
of this section, resulting in recently different karyotypic size (Nishikawa et al. 1984; Roalson et al. 2007; Fig. 12.4).
equilibria within particular clades. This negative 2C/2n correlation could be explained by
If such divergent orthoselection is also common in other chromosomal fission sometimes resulting in minute
species with holocentric chromosomes, it should result in the fragments that are lost because they lack kinetochoric
rapid divergence of chromosome size over a relatively short properties. However, the loss of artificially-induced small
evolutionary time scale, for example, within genera. In fact, fragments has rarely been observed in holocentrics (de
such extensive divergence in chromosome size is clearly Castro et al. 1949). Instead, although no clear explanation
apparent more often in holocentric plant genera compared for the negative 2C/2n relationship has so far been found, it
with monocentric genera (Mann-Whitney, p ¼ 0.0003, is possible that agmatoploidy is accompanied by an unrec-
Fig. 12.3). ognized mechanism of DNA diminution in holocentric
If fission and fusion are the main causes of rapid chromo- organisms. Indeed, if the genome sizes of particular phylo-
somal size divergence in holocentrics, then one might expect genetic lineages of holocentrics are compared with their
that a plot of chromosome number (x-axis) versus genome sister monocentric relatives (Fig. 12.5), in almost all cases,
size (y-axis) would give a straight line parallel to the x-axis holocentrism is associated with the smaller genome size.
12 Holocentric Chromosomes
Fig. 12.4 Genome size versus chromosome number in holocentric
(black; N ¼ 64) and monocentric (grey; N ¼ 3624) angiosperm spe-
cies for which both the genome size and chromosome number are
known (Data taken from the Plant DNA C-values Database, Bennett
and Leitch 2005). For holocentrics, Spearman’s Rho ¼ 0.55; for
An alternative explanation for the observed negative 2C/2n
relationship is that chromosomal fusion (i.e., symploidy) has
been accompanied by retrotransposon proliferation. Indeed, in
the only study of retrotransposons in plants with holocentric
chromosomes Zedek et al. (2010) found that massive
retrotransposon proliferation was responsible for the increased
chromosome sizes in species of genus Eleocharis.19 In fact, the
2C/2n relationship is significantly positive in this genus, at
least in the recently diverged Eleocharis subser. Eleocharis,
where both the largest chromosomes and the highest density of
retrotransposons in the genus are found (Zedek et al. 2010).
12.7.2 Polyploidy
The positive 2C/2n linear correlation detected in Eleocharis
(Zedek et al. 2010) suggests that in addition to agmatoploidy
and symploidy, polyploidy is as important mechanism of
holocentric karyotype evolution. The periodicities in the
chromosome numbers indicate that polyploidy is likely to
be present in several holocentric genera, such as Drosera
(Droseraceae; Rivadavia et al. 2003), Luzula (Juncaceae;
Kirschner 1992, 1995: also confirmed by allozyme analysis),
19
In those species with the largest chromosomes, Ty1-copia LTR
retrotransposons (assuming that all were full-length) might form up to
70% of the genomic DNA (Zedek et al. 2010)
199
monocentrics, Spearman’s Rho ¼ 0.09 (p < 105 for both). The
monocentric palm Voanioala gerardii (Arecaceae; 2n ¼ 596;
2C ¼ 60 pg) is outside of the scatter plot scale but was included in
the test
and Juncus (Juncaceae; Cope and Stace 1985; Rooks 2008:
also confirmed by flow cytometry).
In Cyperaceae, polyploidy (as described above) occurs in
Eleocharis (da Silva et al. 2008b, 2010: autopolyploidy in E.
sellowiana confirmed by FISH by a doubled number of
rDNA sites) and in Rhynchospora (Vanzela et al. 2000,
2003: autopolyploidy in R. tenuis confirmed by rDNA
FISH). Based on the periodicity of prevailing chromosome
numbers, polyploidy is also assumed to occur in Fimbristylis
(Yano and Hoshino 2006), Bulbostylis and Abildgaardia
(Luceño et al. 1998). In Carex, the most specious genus of
Cyperaceae with more than 2,000 species, polyploidy
appears to be surprisingly rare (Hipp 2007; Hipp et al.
2007, 2009; Roalson 2008). The best-documented but rare
example of intraspecific ploidy variation is in Carex
siderosticta, where autotetraploidy was confirmed based on
the formation of tetravalents during meiosis (Tanaka 1940,
1949). The other examples of proven polyploidy in Carex
dolichostachya, C. jackiana, and C. roraimensis are
reviewed in detail by Hipp et al. (2009); while some other
cases are also discussed by Rotreklová et al. (2011). To
explain the lower frequency of polyploidy in this genus,
Heilborn (1934) hypothesized that the formation of
unreduced pollen dyads during pseudomonad pollen devel-
opment may be prevented. During ovular gametogenesis,
three of the four megaspores are aborted, and only one
develops into the ovule sac, whereas during regular pollen
development in most angiosperms, all four microspore
nuclei develop into pollen grains, similar to spermatogenesis
200
Fig. 12.5 Genome size in holocentric plant species (black) compared
with related monocentric plants (grey) (Data taken from the Plant DNA
C-values Database, Bennett and Leitch 2005). (a) Holocentric angio-
sperm species in which the somatic DNA amount is known (N ¼ 79)
have significantly smaller genomes than the remaining putatively
monocentric angiosperm species (N ¼ 4348; Mann Whitney,
p ¼ 0.000; outliers and extremes are not shown in the boxplot but are
included in the test). (b) Holocentrics in the clades of Cyperaceae and
Juncaceae (N ¼ 71) have significantly smaller genomes than the
monocentrics in the sister family Poaceae (N ¼ 470; Mann-Whitney,
p ¼ 0.000). (c) Holocentric Chionographis japonica has a smaller
in animals. In contrast, during pseudomonad pollen meiosis
in Cyperaceae, the degeneration of three of the four nuclei
occurs, similar to megasporogenesis. Among other
angiosperms, this type of pollen development is rare, and
currently only reported in the family Ericaceae (in some
genera of the subfamily Styphelioideae; Furness and Rudall
2010). The remaining question is whether the opportunity
for polyploidy during “regular” pollen and egg development
depends more or exclusively on unreduced pollen produc-
tion, as was implicitly assumed by Heilborn. Recently,
the unreduced egg, not the unreduced pollen, was shown to
be the most likely source for the origin of polyploids
(Krahulcová et al. 2004; Ramsey 2007), even if the natural
frequency of producing non-reduced gametes is similar
during microsporogenesis and megasporogenesis (Ramsey
and Schemske 1998). Moreover, the rare occurrence of
unreduced pollen in Carex podogyna (Tanaka 1941),
C. recta (Cayouette and Morisset 1986a), C. aquatilis
(Cayouette and Morisset 1986b), and Eleocharis mamillata
(Strandhede 1965c) and in the hybrids Carex canescens 
P. Bureš et al.
genome than any other species of the family Melanthiaceae, to which
it belongs (completed with the data from Pellicer et al. 2010; not
tested). (d) Holocentric species in the genus Drosera (N ¼ 6;
Droseraceae) have significantly smaller genomes than the putative
monocentric species in the sister families of Drosophyllaceae,
Nepenthaceae, Tamaricaceae, Plumbaginaceae, and Polygonaceae
(N ¼ 26; Mann-Whitney, p ¼ 0.045). (e) The genome size of the
holokinetic Myristica (N ¼ 2) does not differ from that of the puta-
tively non-holocentric species in the sister families of Magnoliaceae,
Eupomatiaceae, and Annonaceae (N ¼ 26; Mann-Whitney, not
significant)
C. lachenalii (Toivonen 1981) and C. remota 
C. paniculata (Luceño 1994) was clearly shown during
pseudomonad development. The presence of polyploidy in
other Cyperaceae20 (see above) suggests the absence of a
causal relationship between pseudomonad microsporogene-
sis and the low frequency of polyploidy in Carex.
12.7.3 The Enigmatic Case of Luzula: Concerted
Fission and True Polyploidy
In the karyotypes of the holocentric genus Luzula
(Juncaceae), at least three distinct size categories of
chromosomes are recognized: large (AL), medium (BL),
and small (CL), where the relation between their sizes is
20
The pseudomonad pollen formation was shown to be synapomorphic
for the more derived clades of Cyperaceae, but absent in the early
diverging clade of the subfamily Mapanioideae (Simpson et al. 2003).
12 Holocentric Chromosomes
1AL ¼ 2BL ¼ 4CL.21 The somatic chromosome numbers
of particular species are usually regular “euploids” with a
basic chromosome number of x ¼ 6. In the larger size cate-
gory (AL), four regular ploidy levels are present: diploids
(2n ¼ 12 AL; e.g., L. campestris subsp. campestris,
L. pallidula, and L. taurica), tetraploids (2n ¼ 24 AL;
e.g., L. divulgata), hexaploids (2n ¼ 36 AL; e.g., L.
multiflora subsp. frigida) and octoploids (2n ¼ 48 AL;
e.g., L. congesta). In the medium size category, only one
unimodal karyotype, 2n ¼ 24 BL, is known, and it is found
in L. fallax and L. calabra. The same is true in the smallest
category, where 2n ¼ 48 CL is present in L. sudetica. Based
on this pattern, agmatoploidy supposedly operates at the
diploid level, and agmatoploid chromosome sets are then
carried to higher ploidy levels via allopolyploidy
(Nordenski€ old 1961; Kirschner 1992). Support for this so-
called complete agmatoploidy (Luceño and Guerra 1996) or
concerted fission has been obtained from observations of the
meiotic behaviour of chromosomes in crosses between L.
pallescens (2n ¼ 12 AL) and L. sudetica (2n ¼ 48 CL), in
which each of the six large chromosomes (AL) was predom-
inantly associated with four smaller ones (CL; Nordenski€old
1951, 1964). The estimation of DNA content in particular
species also supports the presence of concerted fission (or
concerted fusion) combined with polyploidy (Barlow and
Nevin 1976; Bačič et al. 2007).22
In a few rare cases, Nordenski€ old (1951) found two
cytotypes (fragmented and non-fragmented) within particu-
lar species, such as Luzula spicata, where both the regular
diploid and complete agmatoploid patterns of 2n ¼ 12 AL
and 2n ¼ 24 BL, respectively, were observed. A putatively
similar pattern was also documented in Juncus biglumis
which belongs to the same family as Luzula. Here
Sch€onswetter et al. (2007) found three cytotypes, of which
two, 2n ¼ 60 and 2n ¼ 120, differed an average of just
1.06-fold in their DNA content, suggesting the possibility
of complete agmatoploidy in generating the 2n ¼ 120
21
In addition to these three main size categories, giant (A0) and tiny
chromosomes are present in this genus in Luzula elegans
(¼ L. purpurea) and L. pilosa, respectively. See inner box in Fig. 12.3.
22
An extremely high intraspecific and intraplant variation in chromo-
some number was found by Kuta et al. (2004) in the cultivated
seedlings of Luzula multiflora, in which the somatic chromosome
number varied 6.83-fold (from 12 to 84), whereas the DNA content
varied just 1.04-fold (from 1.796 to 1.864 pg). This observation
suggests a high frequency of agmatoploidy even during ontogenesis;
however, other studies have not documented such chromosome varia-
tion in this species (Nordenski€old 1951; Kirschner 1992; Bačič et al.
2007). Because samples were based on seeds obtained from the Botani-
cal Garden of Stuttgart (Kuta et al. 2004), testing whether that observed
pattern is also present in natural populations or among progeny
cultivated from the seeds collected from wild plants of this species
would be appropriate.
201
karyotype. A descending multiplicative decrease in the num-
ber of chromosomes has also been reported in some phylo-
genetic lineages of butterflies in the family Nymphalidae
which have holocentric chromosomes (Brown et al. 2007),
suggesting an inverted process of concerted fusion.
Although this enigmatic pattern of concerted fission has
been known for almost 60 years, no clear mechanism has
been found to be responsible for the half-breaking of the
chromosomes in Luzula (Greilhuber 1995; Luceño and
Guerra 1996; Madej and Kuta 2001; Guerra et al. 2010).
Roalson et al. (2007) hypothesized that the possible chromo-
somal break points could be the remainders of telomeres
within previously fused chromosomes.23 The typical chro-
mosomal “fragile sites” have been shown to be comprised of
expanded microsatellites or AT-rich minisatellite arrays
(Sutherland et al. 1998) and it is possible that the break
points on Luzula chromosomes are also comprised of satel-
lite repeats possibly related to the LCS1 repeat identified in
Luzula nivea (2n ¼ 12AL) by Haizel et al. (2005) and
shown to be present in at least five sites in each chromosome.
Talbert et al. (2009) speculated that the chromosome
“halves” or “quarters” that result from symmetric chromo-
some breaks during karyotype evolution in Luzula could be
analogous to the developmentally programmed DNA
rearrangements that occur during the ontogenetic cycles of
the round worm Parascaris univalens (Nematoda). In this
holocentric nematode, two chromosomes are present in the
germline cell lineage, whereas somatic cells contain about
60 small chromosomes that originate via fission of the two
large germline chromosomes. Moreover, the amount of
DNA is much lower in the somatic nuclei than in the
germline nuclei because these small fragmented
chromosomes originate from just the central part of the
germline chromosomes. The distal ends of the germline
chromosomes do not form daughter nuclei and degenerate
in the cytoplasm of somatic cells in a process known as
chromatin diminution; a process that has also been detected
in several parasitic nematodes (Pimpinelli and Goday 1989;
Tobler and M€uller 2001). The vast majority of this
eliminated DNA is non-coding, but in rare cases, it contains
a large number of genes (Nemetschke et al. 2010). Interest-
ingly, chromosomal breakpoints in Parascaris do not con-
tain the expected repetitive sequence motifs (Tobler and
M€uller 2001) that are found in the “fragile sites” of
chromosomes (see above). The process of concerted fusion
or fission could be facilitated by some kind of karyotype
orthoselection as proposed by Greilhuber (1995), for exam-
ple, by meiotic drive (see Sect. 12.8).
23
The probable presence of such fragile points on the chromosomes of
Carex has also been speculated by Luceño (1994).
202
12.8 How and Why Holocentric
Chromosomes Originate
The scattered phylogenetic distribution of holocentrism
suggests that it has evolved independently several times
during the course of evolution (Dernburg 2001). However,
some authors theorize that the diffuse centromere is the
ancestral state and that localized centromeres represent a
specialized, derived structure (Mola and Papeschi 2006 and
references therein). This might be true in animals, where
large holocentric clades alternate with monocentric lineages.
In land plants, however, holocentrics are only found among
angiosperms, where they are found in the terminal positions
of phylogenetic trees, they should therefore be considered as
a derived character (Greilhuber 1995). The absence of tran-
sient forms suggests that the change from monocentrism to
holocentrism represents a significant jump similar to “major
evolutionary transitions” (sensu Maynard Smith and
Szathmáry 1995), in which the complexity of chromosomes
is abruptly increased. The exact mechanisms that give rise to
holocentrics are still unknown although it is tempting to
speculate that the origin of holocentric chromosomes is a
very simple process, given that they have arisen indepen-
dently several times during the evolutionary history of
plants.
Even though several hypotheses have been proposed,
none of them have yet been confirmed or disproven. First,
the holocentric state is proposed to arise through the spread
of centromeric sequences from one location to multiple sites
along the chromosomal arms. Such a spread could occur by
the proliferation of transposable elements that carry a cen-
tromeric sequence or by centric fusions and subsequent
pericentromeric inversions (Greilhuber 1995; Diaz et al.
2010). The transposition of retroelements in subtelomeric
regions combined with the modification of the tubulin-based
cytoskeleton was proposed by Villasante et al. (2007) to be
responsible for the origin of primitive centromeres from
telomeres during “major evolutionary transitions” from pro-
karyotic to eukaryotic chromosomes. Such initially unstable
“ditelocentric” chromosomes could subsequently evolve
into chromosomes with a holocentric organization24 by the
continuous spreading of end sequences throughout the chro-
24
Considering that terminal blocks of heterochromatin exhibiting “cen-
tromeric activity” in telokinetic (holocentric) germline chromosomes
of the nematode Parascaris univalens originated by the segmental
amplification of repeats derived from telomeric repeats (Niedermaier
and Moritz 2000).
P. Bureš et al.
mosome25 or to metacentric (monocentric) chromosomes, as
these authors suggested.
Another elegant and simple mechanism for monocentric/
holocentric transition could be that the orthogonal direction
of kinetochore formation against the chromosomal axes has
turned by 90 , allowing the extension of kinetochores along
the chromosome axis (Nagaki et al. 2005). This new kineto-
chore formation would become embedded in the longitudi-
nal groove along the poleward chromatid surface, as is
observed in holocentrics (see Sect. 12.4), rather than develop
into the primary constriction seen in monocentrics. This
putative change in the direction of kinetochore formation is
sometimes considered to be causally related to a lack of
condensin I in holocentric chromosomes and co-localization
of condensin II with CENH3 along the entire chromosomal
length (Ono et al. 2003, 2004; Hauf and Watanabe 2004), but
is more likely to be a consequence, rather than a cause, of
holocentrism (see Sect. 12.5).
Research efforts should now focus on finding common
features among the isolated cases of holocentrism that could
help to elucidate to what extent the transition from
monocentric chromosomes to holocentric chromosomes (or
vice versa) is a general predisposition for all eukaryotes.
However, even if there is a common (epi-)genetic predispo-
sition to holocentrism, an open question remains: What are
holocentric chromosomes good for?
One explanation for the origin of holocentric
chromosomes is based on the mechanics of mitosis and
meiosis (Diaz et al. 2010). During anaphase, mono-
centric chromosomes are pulled toward opposite spindle
poles with the centromere leading and the chromosomal
arms lagging, resulting in “V-shaped” chromosomes. Pulling
chromosomes by one point (centromere) may impose a
mechanical penalty because the chromosomes must be
pulled far enough to ensure that they are not in the way of
the structures that divide the cell during cytokinesis. Indeed,
such a mechanical penalty would be proportional to the
length of the chromosome. In contrast, holocentric chromo-
somes attach spindle microtubules along their entire length
and move to the poles in an orientation that is parallel to the
equatorial plate. Thus, holocentric chromosomes could
make cell division easier, regardless of the length of the
chromosomes (Diaz et al. 2010).
Prevention of deleterious effects of chromosomal fission
or fusion due to the function of the kinetochore along the full
chromosome length seems to be a rather anthropocentric
25
Tandem repeats (including the telomeric repeat TTAGGC) are
indeed dispersed at multiple internal sites of the holocentric
chromosomes of the nematode Caenorhabditis elegans, although they
are more abundant at the ends (C. elegans Sequencing Consortium
1998).
12 Holocentric Chromosomes
explanation because death of an individual due to chromo-
somal breakage usually has no impact on the survival of a
species itself. A theoretical exception might exist if some
agent (e.g., irradiation) generates frequent chromosomal
breakages and thus confers selective advantage to the spe-
cies in which survival is not diminished, i.e., holocentric
relative to monocentric species. Theoretically, fragmenta-
tion would increase if selection favored recombination,
whereas fusion would be the result of selection for reduced
recombination (Stephan 2007). The nature of the pressure
that would select for a certain recombination rate, however,
remains a mystery. In some holocentrics (Caenorhabditis,
Luzula), the crossover rate has been documented to be only
one or two per homologous pair of chromosomes (Monen
et al. 2005; Nordenski€ old 1962). Agmatoploidy might com-
pensate for such lower recombination rates in holocentric
organisms because it leads to an increase in chromosome
numbers and thus also in the number of possible chromo-
somal combinations in zygotes (see Sect. 12.7).
An interesting recent hypothesis suggests that holocentric
chromosomes might have evolved as a defense against the
accumulation of centromeric satellites via “centromere
drive”, a special type of meiotic drive (Talbert et al. 2009).
The centromere drive model, which has only been described
in monocentrics to date, postulates that some homologous
centromere variants can be preferentially transmitted to the
next generation through non-random segregation in asym-
metric meiosis (Henikoff et al. 2001), after which only one of
four meiotic products remains viable and becomes a mega-
spore in plants or an egg in animals. The success of a certain
centromere variant depends on its orientation during the first
meiotic division (Malik and Henikoff 2009). The question
remains as to how a centromere can achieve a preferential
orientation toward one of the poles. To explain this phenom-
enon, the meiotic spindle has been proposed to have func-
tional asymmetry (de Villena and Sapienza 2001a), meaning
that the egg pole and the polar body pole differ in the number
of microtubules emanating from each and consequently, in
their ability to capture centromeres. Thus, the egg pole
captures more or “stronger” centromeres than the polar
body pole or vice versa. Chromosomal fusion, for example,
Robertsonian translocation (the fusion of two acrocentric
chromosomes into one large metacentric chromosome),
reduces the number of centromeres. In human female meio-
sis, Robertsonian chromosomes are preferentially transmit-
ted to the egg over the two non-fused, acrocentric homologs,
whereas the opposite behaviour is observed in mouse females.
In other words, the human egg pole preferentially captures
fewer centromeres, whereas the mouse egg pole captures
more centromeres (de Villena and Sapienza 2001b;
Underkoffler et al. 2005). Similarly, the functional asymmetry
of the meiotic spindle may maintain B chromosomes in a
203
population despite their potentially negative effect on the
fitness of their carriers (Talbert et al. 2009).
The expansion of centromeric satellite repeats can create
a larger (i.e., “stronger”) centromere that recruits more
kinetochore proteins and better attracts microtubules
(Malik and Henikoff 2009; Talbert et al. 2009), which may
provide an advantage or disadvantage, depending on the
preferences of the poles, for this “stronger” centromere in
the competition for the megaspore pole. A centromere with
such an advantage would quickly spread throughout a popu-
lation and might even eventually become fixed. However,
the spreading of the “stronger” centromere within a popula-
tion might be accompanied by a number of negative effects.
Any deleterious mutations carried on the respective chromo-
some would also spread within the population. In addition,
the heterozygosity of the centromere variants could cause
imbalances and non-disjunctions during male meiosis,
which often results in aneuploidy and/or increased male
sterility (Henikoff et al. 2001). In plants, the deleterious
effects of centromere drive caused by the expansion of
centromeric satellites on male fertility were documented in
monkeyflowers (Mimulus; Fishman and Saunders 2008).
Centromere drive may also lead to skewed sex ratios in a
population, as has been shown in birds (Rutkowska and
Badyaev 2008).
Any process or mechanism that suppresses centromere
drive would obviously be favored (Malik and Henikoff
2009).26 A simple switch in the preference of the spindle
poles, for example, from capturing more/“stronger”
centromeres to capturing fewer/“weaker” centromeres,
would function against centromere drive. These switches
have occurred many times during the evolution of mammals
(de Villena and Sapienza 2001c). Mutations in kinetochore
proteins, which interact with centromeric satellite repeats,
can also suppress centromere drive (Henikoff et al. 2001).
The specialized and fundamental function of centromeric
DNA binding proteins, such as CENH3 or CENP-C (centro-
meric protein C), in kinetochore assembly is well-conserved
across eukaryotes. Hence, they would be expected to evolve
under the strong pressure of negative (purifying) selection.
Surprisingly, the reverse is true. The DNA binding domains
of either CENH3 or CENP-C have been reported to have
evolved rapidly in an adaptive manner in every investigated
eukaryotic organism displaying asymmetric meiosis,
26
Actually, this observation would be true in animals, which are usu-
ally gonochoric. In plants, the male sterility of structural heterozygotes
(individuals possessing different types of homologous centromeres)
should not decrease population fitness because these male-sterile
individuals have to produce sexual progeny exclusively via
outcrossing, which would thus counterbalance the eventual inbreeding
depression that originates due to the selfing of hermaphrodites. In other
words, a pattern similar to gynodioecy would result.
204
including plants (Talbert et al. 2004; Malik and Henikoff
2009). The adaptive mutations of CENH3 or CENP-C could
weaken the “stronger” centromeres or reinforce the
“weaker” centromeres, thus restoring balance and sup-
pressing centromere drive (Dawe and Henikoff 2006).
In holocentric chromosomes, the expansion of satellite
repeats seems to have no function because the kinetochores
assemble along the entire chromosome, meaning that there
should not be any “weak” or “strong” centromeres. The
hypothesis proposing that holocentric chromosomes are an
adaptation against the accumulation of centromeric satellites
via “centromere drive” might be supported by the findings in
Luzula nivea. LCS1 was found in arrays that were about
50 kb in size, which is smaller than any other reported
centromeric satellite array in plants (Haizel et al. 2005).
Similarly, Caenorhabditis elegans almost completely lacks
satellite repeats (Emmons 1988; Talbert et al. 2009).
Although the accumulation of centromeric satellites might
be suppressed in holocentrics, centromere drive could still
occur and thus drive variations in chromosome size or num-
ber. For example, in a structural heterozygote that possesses
a larger fused chromosome together with its two non-fused
homologs, the larger chromosome should have a higher or
lower chance to attract spindle microtubules emanating from
the pole that anchors more or fewer microtubules, respec-
tively, in the competition with its non-fused homologs.
Indeed, the ovular pole could emanate more or fewer
microtubules, which would then increase or decrease,
respectively, the chance of the larger chromosome being
transmitted to the next generation. Alternations in such
“holocentric drive” could be the reason for the divergent
karyotype orthoselection in chromosome numbers observed
in Carex sect. Ovales (see Sect. 12.7.1.). Here, as in most
other Cyperaceae, asymmetric meiosis also occurs during
pollen development (see Sect. 12.7.2.). Switching between
the “weak” or “strong” egg poles during “holocentric drive”
could also result in a large variation in chromosomal size,
which is found more often in holocentric genera compared to
monocentric genera (see Sect. 12.7.1, Fig. 12.3).
“Holocentric drive” combined with species range expansion
could also explain the geographic patterns in the intraspe-
cific variation of chromosome number observed in some
sedges (see Sect. 12.7.1.). Finally, the massive
retrotransposon proliferation that resulted in the formation
of large chromosomes during the evolution of the
holocentric genus Eleocharis (Zedek et al. 2010) could
also have been facilitated by the same mechanism.
Acknowledgments Our study was supported by the Czech Science
Foundation (Grant no. GACR206/09/1405) and by the Ministry of
Education, Youth and Sports of the Czech Republic (Grants no.
MSM0021622416, and LC06073).
P. Bureš et al.
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 Karyotype Diversity and Evolutionary Trends
in Angiosperms 13
Hanna Weiss-Schneeweiss and Gerald M. Schneeweiss

Contents 13.1 Introduction

13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Each species has a characteristic chromosome complement,
13.2 Trends in Chromosome Number Evolution . . . . . . . . . . 210
13.2.1 Chromosome Numbers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 its karyotype, which represents the “phenotypic appearance
13.2.2 Dysploidy and Aneuploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 of the somatic chromosomes in contrast to their genic
13.2.3 Polyploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 contents” (Levitzky 1931). In other words, the karyotype is
13.3 Trends in Karyotype Structure Evolution . . . . . . . . . . . . 214 the highest level of structural and functional organization of
13.3.1 Morphological Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 the nuclear genome. By comparing the chromosomes of
13.3.2 Chromosome Banding and Molecular Landmarks . . . . . . 217 different taxa much can be learned about patterns and
13.4 Specialized Chromosomes and Chromosome Systems 221 mechanisms of karyotype evolution and its significance for
13.4.1 Sex Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 diversification and speciation.
13.4.2 Permanent Translocation Heterozygotes . . . . . . . . . . . . . . . . . 222 Karyotype constancy, both in chromosome number and
13.5 Karyotype Evolution in the Genomic Era . . . . . . . . . . . . 222 structure, results from the necessary fidelity of chromosome
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 replication and ensures the faithful transfer of genetic mate-
rial to the next generation. On the other hand, karyotype
variation confers evolutionary change. There are two major
categories of karyotype variation: numerical (resulting usu-
ally from meiotic or mitotic spindle malfunction) and struc-
tural (usually arising from errors in crossing over). These
two major classes are, however, interrelated and the actual
evolutionary change of a karyotype is usually complex,
involving various mechanisms.
Karyotypic features most commonly recorded for compara-
tive evolutionary analyses are chromosome number, size and
symmetry, as well as position and type of chromosomal
landmarks such as centromeres, secondary constrictions, or
repetitive DNA types. These parameters may be complemented
with genome size estimations and data on chromosomal behav-
ior, especially during meiosis. This chapter will describe the
most relevant features and discuss their variation and evolu-
tionary trends within angiosperms, including their role in diver-
sification and speciation.

H. Weiss-Schneeweiss (*)
Department of Systematic and Evolutionary Botany, University
of Vienna, Rennweg 14, Vienna A-1030, Austria
e-mail: hanna.schneeweiss@univie.ac.at

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 209

DOI 10.1007/978-3-7091-1160-4_13, # Springer-Verlag Wien 2013
210
Fig. 13.1 Chromosome number and structure diversity in angiosperms.
(a) Oxalis namaquana/Oxalidaceae: 2n ¼ 2x ¼ 14 (arrowheads indi-
cate secondary constrictions; DAPI staining); (b) Barnardia numidica/
Hyacinthaceae (formerly in genus Scilla): 2n ¼ 2x ¼ 18 (arrowheads
indicate satellited chromosomes); (c) Bellevalia paradoxa/Hyacinthaceae:
2n ¼ 2x ¼ 8; (d) bimodal karyotype of Aloe paralellifolia/
Asphodelaceae: 2n ¼ 2x ¼ 14; (e) Senecio carniolicus/Asteraceae: 2n
¼ 6x ¼ 120; (f) Nectaroscilla hyacinthoides/Hyacinthaceae: 2n ¼ 2x
¼ 20 (arrowheads indicate polymorphic homologous chromosome pair);
13.2 Trends in Chromosome Number
Evolution
13.2.1 Chromosome Numbers
Chromosome number is still the most consistently and fre-
quently recorded cytological character. In part, this is due to
the relative ease of its observation and its nature of being a
discrete character, whereas other karyotypic features, such
as chromosome structure or composition, are technically
more challenging to collect and often more difficult to
quantify.
Chromosome numbers have been determined for about
25–30% of angiosperms (Bennett 1998; Guerra 2008; see
Fig. 13.1 for examples). The taxonomic coverage is, how-
ever, very uneven. In some genera, chromosome numbers
are characterized in detail in many or even all species and
from numerous populations (e.g., Brachypodium/Poaceae:
Watanabe et al. 1999; Crepis/Asteraceae: Babcock 1947;
H. Weiss-Schneeweiss and G.M. Schneeweiss
(g) Melampodium longipilum/Asteraceae: 2n ¼ 2x ¼ 20 (arrowheads
indicate secondary constrictions); (h) Melampodium montanum/
Asteraceae: 2n ¼ 2x ¼ 22 + 5B chromosomes (B chromosomes
encircled); (i) Melampodium mimulifolium/Asteraceae: 2n ¼ 2x ¼ 18
(arrowheads indicate satellited chromosomes); (j) tetraploid
Melampodium paniculatum/Asteraceae 2n ¼ 4x ¼ 36. [All photos H.
Weiss-Schneeweiss (a, with J. Suda, Charles University, Prague)]. Scale
bar represents 5 mm
Dimitrova and Greilhuber 2001; Hypochaeris/Asteraceae:
Stebbins 1971; Cerbah et al. 1998; Weiss-Schneeweiss
et al. 2003, 2007a, 2008; Melampodium/Asteraceae:
Weiss-Schneeweiss et al. 2009; Nicotiana/Solanaceae:
Goodspeed 1933; Narayan 1987; Lim et al. 2000), whereas
other groups remain poorly characterized chromosomally
(e.g., Lentibulariaceae and many tropical plant families).
To ease the navigation through continuously published,
numerous and highly dispersed chromosome number data
several comprehensive chromosome number indices have
been compiled over the years. These include, for example,
Darlington and Wylie (1955), Fedorov (1969), Moore (e.g.,
1974, 1977), Goldblatt and Johnson (1979—; accompanied
by an online version hosted by Missouri Botanical Garden:
http://www.tropicos.org/Project/IPCN). Compilations may
also be available for specific taxa, such as the online Index
to Chromosome Numbers in Asteraceae (http://www.lib.
kobe-u.ac.jp/infolib/meta_pub/G0000003asteraceae_e), or
for certain regions, usually specific countries (e.g., L€ ove
and L€ove 1974; Dobeš and Vitek 2000; Góralski et al. 2009).
13 Karyotype Diversity and Evolutionary Trends in Angiosperms
Known angiosperm chromosome numbers are commonly
reported as sporophytic diploid numbers 2n, but also as
gametic haploid numbers n. For comparative analyses the
(usually inferred) haploid chromosome number of a clade’s
(mostly hypothetical) ancestor is referred to as base chromo-
some number x for the group or clade. This x is not to be
confused with the monoploid number x: the lowest haploid
chromosome number of a polyploid series (Guerra 2008).
Known angiosperm chromosome numbers vary 160-fold.
They range from 2n ¼ 4 (Zingeria biebersteiniana and
Colpodium versicolor/Poaceae, Ornithogalum tenuifolium/
Hyacinthaceae, Brachycome dichromosomatica and
Haplopappus gracilis/Asteraceae: Cremonini 2005;
Rhynchospora tenuis/Cyperaceae: Vanzela et al. 1996) to
2n ¼ c. 600 in the palm Voanioala gerardii (Arecaceae:
oser 1994) and 2n ¼ 640 in the stone-
Johnson et al. 1989; R€
crop Sedum suaveolens (Crassulaceae: Uhl 1978). Chromo-
some numbers of the majority of angiosperms range
from n ¼ 7 to n ¼ 20 (Grant 1982; Masterson 1994).
Chromosome number diversification does not correlate
with morphological diversification, as is evident from mor-
phologically diverse but chromosomally uniform island
radiations (Stuessy and Crawford 1998; Mandáková et al.
2010a) and morphologically uniform, but chromosomally
diverse monocot lineages (Crocus vernus/Iridaceae: Frello
and Heslop-Harrison 2000; Prospero/Hyacinthaceae:
Ainsworth et al. 1983, Fig. 13.2).
The wide range of chromosome numbers is not uniformly
distributed among orders, families and genera of
angiosperms. Some groups show no or very limited chromo-
some number variation (Lilium/Liliaceae: Stewart 1947;
Smyth et al. 1989; Hepatica/Ranunculaceae: Weiss-
Schneeweiss et al. 2007b; Galtonia/Hyacinthaceae: Forrest
and Jong 2004; many woody angiosperm lineages:
Ehrendorfer 1970; Grant 1982), while others are very vari-
able (2n ¼ 6, 8, 10, 12, 16 in Hypochaeris/Asteraceae:
Stebbins 1971; Cerbah et al. 1998; Weiss-Schneeweiss
et al. 2003, 2008; 2n ¼ 18, 20, 22, 24, 28, 36, 40, 46, 48,
54, 56, 60, 66 in Melampodium/Asteraceae: Stuessy 1971;
Weiss-Schneeweiss et al. 2009; 2n ¼ 4, 6, 8, 10, 12, 14, 16,
18, 22, 24, 26, 27, 28, 30, 36 in Brachycome/Asteraceae:
Watanabe et al. 1999). In plant groups where more than one
base chromosome number is present (x ¼ 5, 6, 7 in Lotus/
Fabaceae: Grant 1991; x ¼ 9, 10, 11, 12, 14 in
Melampodium/Asteraceae: Bl€ och et al. 2009; x ¼ 3, 4, 5, 6
in Crepis/Asteraceae: Babcock and Jenkins 1943), the
ancestral base chromosome number might be identified
either as the dominant one and/or the one found in the
most closely related taxa. Nowadays, however, base chro-
mosome numbers are usually inferred within an explicit
(most often molecular) phylogenetic context (Torrell et al.
1999; Mast et al. 2001; Ellison et al. 2006; Enke and
Gemeinholzer 2008; Hipp et al. 2009) using, for instance,
211
Fig. 13.2 Chromosome number and structure variation in a group of
closely related species and cytotypes in Prospero/Hyacinthaceae (for-
merly in genus Scilla; asterisks indicate large metacentric “fusion” chro-
mosome, where present). (a) P. obtusifolium: 2n ¼ 2x ¼ 8 (arrowheads
indicate secondary constrictions); (b) P. hanburyi: 2n ¼ 2x ¼ 14
(arrowheads indicate satellited chromosomes); (c) P. autumnale s. l.
cytotype B6B6: 2n ¼ 2x ¼ 12 (arrowheads indicate secondary
constrictions); (d) P. autumnale s. l. cytotype B7B7: 2n ¼ 2x ¼ 14
(arrowheads indicate secondary constrictions); (e) P. autumnale
s. l. cytotype AA: 2n ¼ 2x ¼ 14; (f) P. autumnale s. l. cytotype
B7B7: 2n ¼ 2x ¼ 14 + 2B chromosomes (arrowheads indicate
B chromosomes); (g) P. autumnale s. l. cytotype B7B7: 2n ¼ 2x ¼ 14
(arrowhead indicates supernumerary segment in one of the
homologs of chromosome 1); (h) P. autumnale s. l., diploid homoploid
hybrid B6B7: 2n ¼ 2x ¼ 13; (i) P. autumnale s. l. triploid hybrid
B6B6B7: 2n ¼ 3x ¼ 19. (All photos T.-S. Jang and H. Weiss-
Schneeweiss). Scale bar represents 5 mm
maximum parsimony or likelihood based models of chromo-
some number reconstruction as implemented in software
such as Mesquite (Maddison and Maddison 2009) or
chromEvol (Mayrose et al. 2010).
Inferences of chromosome base numbers are becoming
increasingly difficult (or even impossible) with increasing
phylogenetic depth as identical chromosome base numbers
will occur in unrelated lineages, already within a single
genus (e.g., Cerbah et al. 1998; Enke and Gemeinholzer
2008). Additionally, karyotype rearrangements (with or
without polyploidy: see Sect. 13.2.3) as major drivers of
stepwise chromosome number changes (see Sect. 13.2.2)
might occur frequently and often rapidly on an evolutionary
time scale (Brassicaceae: Lysák et al. 2006; Prospero/
Hyacinthaceae: Ainsworth et al. 1983; Vaughan et al.
1997). This also affects efforts to determine the ancestral
basic chromosome number of angiosperms. Consequently,
both early estimates by Raven (1975) of x ¼ 7 as well as
more recent reconstructions of x ¼ 6 by Soltis et al. (2005)
need to be viewed with caution. Evidently, more in-depth
karyotypic information for identification of chromosomal
“building blocks” (Schranz et al. 2006) as well as more
212
realistic chromosomal evolutionary models (Dobigny et al.
2004; White et al. 2010) will be necessary for reliable
chromosome number reconstructions.
For a long time the dogma was that a species possesses a
single chromosome number and ploidy level (as well as one
karyotype structure), because deviating somatic chromo-
some numbers usually confer meiotic irregularities or steril-
ity (Stebbins 1971; Levin 2002). In recent years, however,
several plant species have been identified that possess more
than one diploid somatic chromosome number. This phe-
nomenon is most frequently caused by the occurrence of
intraspecific polyploidy (Ehrendorfer 1958; Frello and
Heslop-Harrison 2000; Stuessy et al. 2004; Suda et al.
2007; see also Sect. 13.2.3 and Husband et al. 2013, this
volume). In other species complexes, it entails the presence
of a dysploid series at the diploid level, which may later be
accompanied by hybridization and polyploidy creating com-
plex patterns of chromosome numbers. Examples include
Scilla (Barnardia) scilloides/Hyacinthaceae (2n ¼ 16, 18,
26, 27, 34, 35, 36, 43: Haga and Noda 1976; Choi et al.
2008) or Prospero autumnale/Hyacinthaceae (2n ¼ 10, 12,
14, 25, 26, 27, 28, 42 and so on: Ainsworth et al. 1983; Ebert
et al. 1996). Such intraspecific numerical and/or structural
(see Sect. 13.3) karyotypic variants are called cytotypes or
chromosomal races/cytoraces (Stebbins 1971; Stuessy 2009;
Fig. 13.2) and may in fact constitute cryptic species.
13.2.2 Dysploidy and Aneuploidy
13.2.2.1 Dysploidy
Dysploidy refers to the stepwise change of the haploid chro-
mosome number among related species (Stebbins 1971;
Guerra 2008) and such a series of haploid chromosome
numbers constitutes a dysploid series (Ehrendorfer 1964).
Although chromosome number change may involve the loss
or gain of entire chromosomes (aneuploidy; Guerra 2008),
much more frequently it is achieved via genome restructur-
ing resulting in centromere/chromosome number reduction
or increase with or without significant loss of genetic mate-
rial (unbalanced and balanced rearrangements, respectively;
see Lysák and Schubert 2013, this volume). A relatively
simple example of balanced rearrangement is centric
fusion-fission (“Robertsonian translocation” in the broad
sense: Guerra 2008). Here, the karyotype involved in a
centric fusion or fission has one chromosome fewer or
more, respectively, while retaining an intact number of
chromosomal arms. Robertsonian fission/fusion events
occur regularly in animals (Imai 1978; Rowell et al. 2002)
including apes and humans (Yunis and Prakash 1982),
and are also documented in some angiosperm groups
(reviewed in Jones 1977, 1998; e.g., Nothoscordum/
Alliaceae: Souza et al. 2009; Gibasis/Commelinaceae:
H. Weiss-Schneeweiss and G.M. Schneeweiss
Kenton 1981; Tradescantia/Commelinaceae: Jones 1998;
Christensonella/Orchidaceae: Koehler et al. 2008; Moreas
et al. 2012). Recent data, however, suggest that in the majority
of angiosperms dysploid changes are more often achieved via
complex mechanisms involving (usually) pericentric inversions
and reciprocal (unequal) translocations acting in concert
(Schubert 2007; for details on mechanisms see Lysák and
Schubert 2013, this volume). This has been elegantly shown
in Brassicaceae (Lysák et al. 2006) and has been suggested for
Fabaceae (Choi et al. 2004) and grasses (Devos 2005).
The majority of earlier studies considered descending
dysploidy (i.e., chromosome number reduction) as the
main trend of dysploid chromosome number change in
angiosperms (Jones 1977), often accompanied by the evolu-
tion of an annual life form (Stebbins 1971). Examples
include x ¼ 6 ! 3 in Crepis/Asteraceae (Babcock 1947),
x ¼ 9 ! 2 in Haplopappus/Asteraceae (Jackson 1962;
Anderson et al. 1974) and x ¼ 15 ! 12 in Crocus/Iridaceae
(Brighton 1978). For some plant groups, ascending
dysploidy (i.e., chromosome number increase) has also
been inferred (x ¼ 7 ! 9 in Allium/Alliaceae: Levan
1932). These largely intuitive inferences might, however,
turn out to be oversimplified. For instance, recent molecular
phylogenetic analyses of Crepis show that chromosome
numbers are largely uncorrelated with phylogeny, and that
there is no simple descending dysploidy (Enke and
Gemeinholzer 2008) as suggested previously. Therefore,
the direction of dysploid chromosomal changes not only is
largely group specific, but often bidirectional with both
types of dysploidy (ascending and descending) co-occurring
within a given group (mixed dysploidy; Bakker et al. 2000;
Barber et al. 2000; Samuel et al. 2003; Enke and
Gemeinholzer 2008; Bl€och et al. 2009). Indeed, mechanisms
which can lead to simultaneous bidirectional dysploid
changes have been previously proposed (Schubert and
Rieger 1985). Irrespective of the direction, dysploid chro-
mosome number changes clearly significantly contribute to
angiosperm diversification and speciation (Stebbins 1971;
Grant 1981; Levin 2002).
13.2.2.2 Aneuploidy
In contrast to dysploidy, aneuploidy refers to the loss or gain
of entire chromosomes (Guerra 2008). As aneuploid varia-
tion significantly changes the genetic balance of an organ-
ism, it is usually disadvantageous or even lethal for its
carrier and thus of little relevance on an evolutionary time-
scale (in contrast to dysploidy; Ehrendorfer 1964, 1980).
Exceptions do, however, exist. The most impressive one is
Claytonia virginica (Portulacaceae) that shows a nearly con-
tinuous series from 2n ¼ 12 up to at least 2n ¼ 100 (occa-
sionally up to 2n ¼ 191) with x ¼ 6, 7, 8 (Rothwell and
Kump 1965; Lewis et al. 1967). Intrapopulational chromo-
somal variation was shown to manifest itself not only in
13 Karyotype Diversity and Evolutionary Trends in Angiosperms
chromosome number variation, but also in plants with the
same chromosome numbers but occasionally with varied
karyotype structures. In addition, different organs of the
same plants were shown to possess different chromosome
numbers (aneusomaty), and climatically driven seasonal
variation was also reported (Lewis et al. 1967). This tremen-
dous chromosome variation in C. virginica is considered to
be the result of hybridization and polyploidization between
partly isolated diploid populations with different haploid
chromosome numbers and subsequent meiotic irregularities,
and/or of ecological factors (Lewis et al. 1967).
In general, the extent of genetic imbalance imposed by
aneuploidy is lower in polyploids. Consequently, aneuploidy
might be expected to be more frequently fixed in polyploids,
resulting in its higher evolutionary potential in a polyploid
background (Lewis et al. 1967; Ainsworth et al. 1983;
Weiss-Schneeweiss et al. 2009). It is however, not always
easy to distinguish dysploidy from aneuploidy in polyploids.
13.2.3 Polyploidy
Polyploidy refers to the instantaneous multiplication of
entire chromosome sets (Stebbins 1971; Wendel 2000;
Leitch and Leitch 2008). The most widely used classifica-
tion concerns the distinction of auto- and allopolyploids
(Kihara and Ono 1927). Whereas in autopolyploids the
(nearly) identical chromosomes within a single (sub)species
are multiplied (Ramsey and Schemske 1998, 2002), allo-
polyploids are the result of hybridization between two
(sub)species accompanied by polyploidization (Stebbins
1971). An alternative to this taxonomic definition has been
the use of polysomic vs. disomic segregation (accompanied
by multivalent vs. bivalent formation in meiosis) suggesting
autopolyploid and allopolyploid origin, respectively
(Rieseberg and Doyle 1989; Doyle et al. 2008; but see also
Stebbins 1971). From the standpoint of chromosome behav-
ior, genetic allopolyploids should behave like diploids with
more sets of chromosomes, showing bivalent formation and
disomic segregation. Both types of definitions are useful and
usually lead to a congruent conclusion, but exceptions do
exist. These include, for instance, allopolyploids, that though
disomic, may exhibit multivalent formation in early stages
after formation (Ownbey 1950; Ramsey and Schemske
2002) or autopolyploids that undergo meiosis with no chro-
mosomal irregularities (Weiss and Małuszyńska 2000). The
difficulties might in fact be more pronounced since the
polysomic condition is usually considered to be an initial
and transient stage leading to chromosomal diploidization
and disomy. Thus, old polyploids with disomic behaviour
might also have originated as autopolyploids (Doyle et al.
2008). Yet another type of polyploid classification is based
on the polyploid’s age and point of origin in the evolutionary
213
history, recognizing (in order of decreasing age) paleo-,
meso- and neopolyploids (Ehrendorfer 1980; Ramsey and
Schemske 1998, 2002; Comai 2005).
The majority of well-analysed polyploids are
allopolyploids. Allopolyploidy might result in a novel chro-
mosome base number, if taxa with different chromosome
base numbers are involved as parents (dibasic polyploidy;
e.g., Brassica/Brassicaceae: U 1935). Contrary to earlier
views (Stebbins 1950, but see Darlington 1956), where
autopolyploids were conventionally treated as intraspecific
cytotypes of a lower ploidy taxon, they are now increasingly
recognized as being equally important for speciation as
allopolyploids (Soltis et al. 2007).
The propensity for polyploidization appears to be
unequally distributed among angiosperms (46% of infra-
generic polyploidy in higher monocots vs. 32% in rosids:
Wood et al. 2009; scarce polyploidy in woody angiosperms
vs. frequent, likely ancient, polyploidy in tropical humid
forests: Ehrendorfer 1970). The underlying causes remain,
however, unknown mostly due to the lack of adequate, in-
depth genetic and genomic information on the extent of
polyploid ancestry.
In contrast to dysploidy, polyploidization is an essentially
unidirectional process towards higher ploidy levels (often
resulting in polyploid series or polyploid complexes;
Stebbins 1940, 1971), a feature termed “polyploidy ratchet”
(Meyers and Levin 2006). Whereas early estimates indicated
that 30% of flowering plant species underwent at least one
round of polyploidization (Stebbins 1950), current estimates
suggest that all angiosperms are ancient polyploids
(paleopolyploids, Jiao et al. 2011) being affected by at
least two ancestral whole genome duplications (WGD;
Bowers et al. 2003; Jaillon et al. 2007; Fawcett et al. 2009;
Jiao et al. 2011). These WGDs have been suggested to play
causative roles for the evolutionary success of angiosperms
(Van de Peer et al. 2009; Jiao et al. 2011). Additional WGDs
have been inferred for specific angiosperm lineages (Schranz
and Mitchell-Olds 2006; Jaillon et al. 2007; Proost et al.
2011). Recent years have provided the wealth of data
demonstrating that polyploidy not only is frequent, but is
one of the major forces in angiosperm diversification and
speciation. Estimates of the frequency of polyploid specia-
tion (speciation events accompanied by ploidy level
increase) range from 3.8% (Levin and Wilson 1976), 2–4%
(Otto and Whitton 2000) to 15% (Wood et al. 2009) (see
Fawcett et al. 2013, this volume).
Polyploidization is a recurrent phenomenon in many
angiosperm lineages (Soltis and Soltis 1999), with numerous
examples of both autopolyploids (Suda et al. 2007;
Trávnı́ček et al. 2011) and allopolyploids (Dixon et al.
2009; Symonds et al. 2010). Consequently, Soltis et al.
(2009) state: The question is no longer “What proportion
of angiosperms are polyploid?”, but “How many episodes of
214
polyploidy characterize any given lineage?” As indepen-
dently originated lineages might apparently show different
evolutionary trajectories (Doyle et al. 2004) and constitute
separate species, the contribution of polyploidy to speciation
might well be underestimated by current taxonomic practice
(Soltis et al. 2010).
The most obvious effect of polyploidization on the kar-
yotype is the increase in chromosome number. Conse-
quently, reproductive isolation conferred by meiotic
problems in hybrids between polyploids and their lower-
ploidy ancestors has long been identified as an important
mechanism leading to polyploid speciation. Polyploi-
dization, often coupled with hybridization does, however,
also trigger a cascade of processes operating at the genomic,
epigenomic and proteomic level (Leitch and Leitch 2008;
Fawcett et al. 2013, this volume). These include activation of
transposable elements, epigenetic phenomena leading to
heterochromatin formation, and eventually accumulation of
structural chromosomal mutations (e.g., via interhomeologous
translocations; Clarkson et al. 2005; Shoemaker et al. 2006;
Doyle et al. 2008), which ultimately lead to complete genome
diploidization (Wendel 2000; Doyle et al. 2008; Mandáková
et al. 2010b).
Genetic and cytological genome diploidization of
polyploids is often accompanied by a reduction in genome
size (Leitch and Bennett 2004; Pellicer et al. 2010) and over
evolutionary time, chromosome numbers of polyploids
might also be considerably reduced, even below the original
level of the ancestral diploid taxa (Lysák et al. 2006). There-
fore, assessment of the incidence of polyploidy from known
chromosome numbers alone will be biased downwards. Such
cycles of revolutionary (polyploidization) and evolutionary
(cytological diploidization) phases have so far only been
well documented for a few angiosperm groups, for which
enough genomic data are available, including Brassicaceae
(Lysák et al. 2006; Mandáková and Lysák 2008), Oryza/
Poaceae (Wang et al. 2007) or Gossypium/Malvaceae (Shoe-
maker et al. 2006).
13.3 Trends in Karyotype Structure Evolution
Whereas data on chromosome numbers accumulate rela-
tively quickly, detailed karyotype information has been
obtained at a much slower pace. Karyotype features detect-
able in classically stained material (e.g., by Feulgen method
or by acetocarmine; Fukui and Nakayama 1996) include, in
addition to chromosome number, individual chromosome
sizes (absolute and relative), haploid (or diploid) karyotype
length, karyotype symmetry, and the positions of morpho-
logically discernible chromosome landmarks, most notably
primary and secondary constrictions. The application of
more advanced staining techniques, including different
H. Weiss-Schneeweiss and G.M. Schneeweiss
banding techniques (C-banding or fluorescent banding:
Deumling and Greilhuber 1982; Greilhuber 1982, 1995;
Lukaszewski and Gustafson 1983) and, recently and most
prominently, fluorescence in situ hybridization (Findley
et al. 2010), provides many more chromosomal landmarks.
These may enable identification of individual (homologous)
chromosomes/chromosome arms in a given karyotype or
even the ‘painting’ of all chromosomes (Lysák et al. 2006).
However, in contrast to the majority of animals, the ability to
label whole chromosomes using specific chromosome
‘paints’ in angiosperms is difficult, mostly due to the high
content of dispersed transposable elements across the
genome and their relatively quick turnover (Lim et al.
2007a). The karyotype and its features can be schematically
represented by an idiogram or karyogram.
13.3.1 Morphological Features
13.3.1.1 Chromosome Size
Individual chromosome sizes in angiosperms (given as mean
chromosome length per species) have been estimated to vary
from 0.6 to more than 14.6 mm, and the total chromosome
length per diploid genome from 14.6 to 250 mm (Levin and
Funderburg 1979). Even within a single karyotype of a taxon
a relatively wide range of chromosome sizes can occur, with
individual chromosome pairs ranging from 1 to 2 mm to
more than 10 mm (Schubert 2007; Hamouche et al. 2010).
Despite such variation, most plant species possess relatively
uniform small to medium-sized chromosomes (c. 1–3 mm;
Stace 2000). Possibly the smallest angiosperm chromosomes
known so far are those in Genlisea/Lentibulariaceae (“bac-
terial size chromosomes”, Greilhuber et al. 2006), which
also holds the record for the lowest genome size. The largest
chromosomes are found in plants with very large genomes,
but not many chromosomes (Lilium/Liliaceae, 2n ¼ 24,
20 mm: Peruzzi et al. 2009; Paris/Melanthiaceae, 2n ¼ 40,
some chromosomes larger than 30 mm: Pellicer et al. 2010).
Chromosome size is limited in both directions. The upper
limit is determined by the length of the longest chromosome
arm. This cannot exceed half of the average spindle axis length
at telophase so as not to impair chromatid separation during
mitosis (Schubert and Oud 1997; Schubert 2001; Hudakova
et al. 2002). The lower limit is more difficult to define. As
chromosomes smaller than 1% of the genome fail to segregate
correctly during meiosis, even if supposedly fully intact with
an original centromere, telomeres and replication origins
(Schubert 2001; Murata et al. 2006), the lower limit appears
to be determined by bivalent stability and chiasma formation
allowing stable transmission during meiosis. Changes in chro-
mosome size can be correlated with changes in genome size
(achieved via polyploidy and/or repetitive DNA amplification;
see below) or might result from interchromosomal
13 Karyotype Diversity and Evolutionary Trends in Angiosperms
rearrangements, such as centric fusions/fissions or unequal
translocations (see Lysák and Schubert 2013, this volume).
13.3.1.2 Karyotype Length and Genome Size
Genome size variation rarely reflects chromosome number
variation because the major mechanisms responsible for
changes in genome size are different from those leading to
chromosome number change. This is best illustrated by the
fact that the species with the smallest genome size, found in
Genlisea (Lentibulariaceae; Greilhuber et al. 2006), have
similar chromosome numbers (i.e., 2n ¼ c. 40) to the spe-
cies with the largest genome size, Paris japonica
(Melanthiaceae; 2n ¼ 40: Pellicer et al. 2010). Total karyo-
type length is correlated with genome size because both are
shaped by the same processes. These include polyploi-
dization and accumulation of repetitive DNA, most impor-
tantly retroelements (Bennetzen 2000; Tenaillon et al.
2011), or DNA deletions via unequal and illegitimate recom-
bination (Bennetzen 2002; Hawkins et al. 2008). Changes in
genome size are best reflected in changes in the size (or more
accurately volume and composition) of chromosomes.
Genome size changes can affect the entire karyotype uni-
formly (Ainsworth et al. 1983; Raina and Rees 1983) or they
may be restricted to a subset of chromosomes usually via
amplification/deletion of blocks of certain families of tan-
dem repeats (de la Herrán et al. 2001; Koo and Jiang 2008).
For more information on genome size evolution in
angiosperms see Leitch and Leitch (2013, this volume).
13.3.1.3 Centromere Position, Holocentric
Chromosomes
In mitotic chromosomes, the centromere appears as a cyto-
logically visible primary constriction and represents the site
for kinetochore assembly and spindle attachment thereby
ensuring faithful segregation of chromosomes during cell
division. Most angiosperms possess chromosomes with
localized centromeres (monocentric chromosomes), which
divide a chromosome into two arms.
Centromere position and consequently arm length ratios
(long arm length/short arm length) have been used as criteria
in the two most commonly used systems of chromosome
nomenclature (Levan et al. 1964; Stebbins 1971; Guerra
1986). Levan et al. (1964) distinguished six chromosome
types (arm ratio in square brackets): M (median point; [1]),
m (median region; [>1–1.7]), sm (submedian region;
[>1.7–3.0]), st (subterminal region; [>3.0–7.0]), t (terminal
region; [>7.0–infinity]), and T (terminal point; [infinity]). In
contrast, Stebbins (1971), modified by Guerra (1986), distin-
guished metacentric [1–1.49], submetacentric [1.50–2.99],
acrocentric [3.00–infinity] and telocentric [infinity]
chromosomes.
Mechanisms leading to shifts in centromere position
include structural rearrangements (pericentric inversions
215
and translocations; see Lysák and Schubert 2013, this vol-
ume) as well as centromere deactivation and de novo centro-
mere formation (Han et al. 2006). Indeed, studies have
shown that the presence of centromere-specific tandem
repeats are neither necessary nor sufficient to form a func-
tional centromere (Nasuda et al. 2005). A fully functional
centromere might be deactivated despite retaining the
centromere-specific repeats (for example in dicentric
chromosomes; Han et al. 2006) and a functional centromere
can be formed de novo in an acentric chromosome fragment
in the absence of specific centromeric repeats (Topp et al.
2009; Guerra et al. 2010).
In contrast to this, in a few genera and species,
centromeres are not localized at specific centromeric
constrictions on the chromosomes. Instead they are dispersed
over the entire chromosome length. Such holocentric or
holokinetic chromosomes (Guerra 2008; Guerra et al. 2010)
are found in a few angiosperm lineages, including
Cyperaceae (Carex, Rhynchospora, Eleocharis; Luceño
et al. 1998; Vanzela et al. 2000; da Silva et al. 2005; Hipp
2007; Zedek et al. 2010) or Convolvulaceae (a single dodder/
Cuscuta species: Pazy and Plitmann 1995; Guerra and Garcı́a
2004), where they are often associated with a specialized
form of inverted meiosis (Greilhuber 1995). The lack of a
localized centromere not only reduces the number of chro-
mosomal landmarks, but may also affect karyotype dynam-
ics. Specifically it has been suggested that chromosome
number change (i.e., dysploidy) can occur rapidly in plants
with holocentrics, since chromosome number change due to
fragmentation (agmatoploidy/agmatopolyploidy) or fusion
(symploidy) is not expected to have deleterious effects.
Such changes have been documented in natural populations
(in Luzula/Juncaceae: Kuta et al. 2004; Carex/Cyperaceae:
Chung et al. 2011), and callus tissue culture (Madej and Kuta
2001). The dynamics of holocentric chromosome number
changes can also involve polyploidy (Luzula/Juncaceae:
Kuta et al. 2004; Juncus bigumis/Juncaceae: Sch€onswetter
et al. 2007; note that the presence of holocentric species in
Juncus is still contentious: Bailey cited in Stace 2000) and
“standard” dysploidy (Guerra 2008; Chung et al. 2011).
Furthermore, their chromatin organization and genome size
dynamics appear to be little influenced by centromeric orga-
nization (Guerra and Garcı́a 2004; Haizel et al. 2005; Zedek
et al. 2010). For a detailed account of holocentric
chromosomes see Bureš et al. (2013, this volume).
13.3.1.4 Karyotype Symmetry, Bimodal
Karyotypes
Karyotype symmetry is determined by the size of the
chromosomes and/or centromere position. Most
angiosperms have similarly sized mostly meta- and submeta-
centric chromosomes, and consequently possess uniform,
symmetric karyotypes (Levitzky 1931; Stebbins 1971;
216
Fig. 13.1). However, some lineages have karyotypes com-
prising chromosomes with a range of sizes and centromere
positions giving rise to asymmetric karyotypes. The majority
of these are found among monocots with larger
chromosomes and genomes (Stedje 1989; Choi et al. 2008;
Hamouche et al. 2010), but they also occur in other groups
with otherwise relatively uniform karyotypes (Babcock
1947; Cerbah et al. 1998). Changes from symmetric to
asymmetric karyotypes most often involve changes in chro-
mosome morphology (from metacetric to telocentric
chromosomes), sometimes accompanied by dysploidy and/
or non-uniform chromosome size changes (Stebbins 1971;
de la Herrán et al. 2001).
Various indices have been suggested to describe karyo-
type asymmetry (reviewed and extended in Paszko 2006).
Interest in these indices, which can serve as concise
descriptors of several karyotype features, also results from
attempts to identify evolutionary trends in karyotype evolu-
tion. Specifically, Stebbins (1971), following Levitzky’s
principle of karyotype symmetry vs. asymmetry (Levitzky
1931), postulated that asymmetric karyotypes are derived
and evolutionarily younger than symmetric ones, because
asymmetric karyotypes have often been found in plants with
specialized morphological features or in evolutionary youn-
ger derivatives. A potential explanation is provided by the
minimum interaction hypothesis (Imai et al. 1986), which
states that karyotype evolution has been mostly shaped by
selection to reduce the occurrence of fitness-reducing chro-
mosomal mutations, such as reciprocal translocations. In
species with big genome size to nuclear volume ratios, this
can be achieved by an increasing chromosome number, e.g.,
by centric fission, leading to acrocentric chromosomes
(Brighton 1978). To our knowledge, the pattern expected
under this hypothesis, i.e., a positive correlation between
karyotype asymmetry and the genome size to nuclear volume
ratio, has never been rigorously tested in plants. The generality
of the derived nature of an asymmetric karyotype has, how-
ever, been questioned (Jones 1978; Dimitrova and Greilhuber
2000). The main criticism is that it is difficult to assess the
direction of such karyotype evolution as many reversals of the
main trend might have occurred (Stace 2000). Recent analyses
in Brassicaceae actually suggest that karyotype asymmetry
might be a transitory state rather than simply a derived evolu-
tionary endpoint (Lysák et al. 2006; Mandáková and Lysák
2008). More data of this type (i.e., detailed information on
chromosomal restructuring studied within an explicit phylo-
genetic context) will be necessary to test general hypotheses
on evolutionary trends in karyotype asymmetry.
A special case of asymmetry is the bimodal karyotype,
which is characterized by the presence of two sharply dis-
tinct size classes of chromosomes, without any gradual tran-
sition. Bimodal karyotypes are well known from monocots,
especially in Asphodelaceae (Aloe: Fig. 13.1d, Haworthia,
H. Weiss-Schneeweiss and G.M. Schneeweiss
Gasteria: Brandham 1971; Brandham and Doherty 1998;
Vosa 2005) and Agavaceae (Agave, Yucca: Vosa 2005;
Robert et al. 2008). Various hypotheses have been proposed
to explain the origin of bimodal karyotypes including the
suggestion that they are derived from the symmetrical
karyotypes of polyploids with the small chromosomes
representing the products of differential loss of chromosome
segments (Darlington 1963). Alternative hypotheses suggest
that bimodal karyotypes are the result of periodical unequal
translocations (Stebbins 1971) or the unequal selective
amplification of certain types of heterochromatin within a
subset of chromosomes (de la Herrán et al. 2001).
13.3.1.5 Secondary Constrictions
In addition to primary constrictions, one or more
chromosomes may also possess secondary constrictions.
These can be located interstitially or, more often,
subterminally, whereby a more or less spherical terminal
chromosome piece called the satellite is segmented off
from the chromosome arm (Figs. 13.1 and 13.2). Secondary
constrictions are the sites of origin of nucleoli (NORs,
nucleolar organizing regions) harboring up to thousands of
tandemly arranged arrays of 35S (18S-5.8S-25/28S) ribo-
somal RNA (rRNA) genes (Volkov et al. 2004) and their
spacers. The rRNA genes are found in all living organisms
(“house-keeping genes”) and code for a subset of rRNA for
building the large and small ribosome units (Schwarzacher
and Wachtler 1986). Active NORs can be detected either by
the presence of secondary constrictions and/or satellites or
by silver-staining, which detects proteins associated with
active NOR regions (Schubert et al. 1979). Detection of all
(both active and inactive) NORs in the genome requires
fluorescence in situ hybridization techniques, and will be
discussed in Sect. 13.3.2.2.
The number of morphologically detectable NORs (active
NORs) may vary greatly among plant groups (reviewed in
Małuszyńska et al. 1998) and to a lesser extent between
closely related species (Kamstra et al. 1997; Cerbah et al.
1998), but rarely within species (Phaseolus vulgaris/
Fabaceae: Pedrosa-Harand et al. 2006; Alstroemeria
pelegrina/Alstroemeriaceae: Baeza et al. 2007). One promi-
nent trend in angiosperms is reduction of the number of
NORs after polyploidization as a result of deactivation or
loss of loci (see also Sect. 13.3.2.2).
13.3.1.6 Supernumerary Chromosomal Material
Karyotypes of many species include supernumerary genetic
material that by definition is not part of a taxon’s regular
karyotype. If physically integrated into the regular karyotype
(i.e., the A-chromosomes) often appearing as a polymorphic
block(s), this material is referred to as a supernumerary
segment (SS; John and Miklos 1979). Supernumerary
segments can be identified in homologous chromosomes in
13 Karyotype Diversity and Evolutionary Trends in Angiosperms
the heterozygous condition due to chromosome length
differences. Although SSs are particularly frequent in some
insect groups (locusts and grasshoppers; John and Miklos
1979), so far they have only been identified in a few plant
groups, particularly in Hyacinthaceae (Greilhuber and
Speta 1978; Ruiz Rejón and Oliver 1981; Garrido-Ramos
et al. 1998; Weiss-Schneeweiss et al. 2004; Fig. 13.2g)
or the chromosomally variable Rumex acetosa group
(Polygonaceae; Wilby and Parker 1988), although it is likely
that they are much more common (J. Parker, pers. comm.).
In most taxa, SSs are heterochromatic (SSH; Ruiz Rejón and
Oliver 1981; Weiss-Schneeweiss et al. 2004), and it is only
in a few cases that they have been shown to be euchromatic
(SSE; Ainsworth et al. 1983; Ebert et al. 1996; Fig. 13.2g).
Regardless of the type, SSs can be located either interstitially
or terminally within the chromosomes. They appear to be
fixed and of ancient origin, often transgressing cytotype,
population, or even species boundaries (Ainsworth et al.
1983; J. Parker, pers. comm.). One of the hypotheses
concerning the origin of SSs suggests that these segments
are relics of chromosome material lost from many, but not
all populations (tolerated deletions; Camacho and Cabrero
1987). Alternatively, SSs might represent novel, selectively
amplified and maintained segments of the genome
containing, for instance, satellite DNA (Shibata et al. 2000)
or heterochromatic segments resulting from unequal
crossing-over (Smith 1976). The role of SSs in karyotype
evolution remains elusive (Wilby and Parker 1988).
If supernumerous genetic material is present as separate
chromosomes these are referred to as B chromosomes (acces-
sory or supernumerary chromosomes; Joneset al. 2008;
Figs. 13.1h and 13.2f). Characteristics of B chromosomes are
(1) lack of recombination with A chromosomes, (2) non-
Mendelian and irregular inheritance resulting in fluctuating
numbers (including nil), and (3) evolutionary trajectories inde-
pendent of those of the A chromosomes. B chromosomes are
usually smaller than A chromosomes (at least than the largest
A chromosomes; Lewis 1951; Żuk 1969; Ainsworth et al.
1983; Parker et al. 1991) and in some cases they are so small
that it is impossible to indentify chromosome arms via light
microscopy (microchromosomes; e.g., in Hypochaeris
maculata/Asteraceae: Parker 1976). In overall structure,
B chromosomes are similar to A chromosomes (being acro-
centric to metacentric in type, sometimes variable within the
same species: Ruiz Rejón et al. 1980; Guillén and Ruiz Rejón
1984). B chromosomes are typically thought to possess no or
very few genes (such as rRNA genes: Małuszyńska and
Schweizer 1989; Dhar et al. 2002; genes coding genetic infor-
mation for their own transmission: Jones et al. 2008). It is,
however, not clear whether any genes that are present are
transcriptionally active (Jones et al. 2008) (see also chapter
by Houben et al. 2013, this volume).
217
It is now widely accepted that B chromosomes have mul-
tiple origins. Most commonly, B chromosomes are considered
to have originated from A chromosomes following chromo-
some number reductions, especially via unequal
translocations (Crepis capillaris/Asteraceae: Jones and Rees
1982); from genomic rearrangements of trisomics (Plantago
lagopus/Plantaginaceae: Dhar et al. 2002); from spontaneous
generation following genomic rearrangements after interspe-
cific hybridization (Coix/Poaceae: Sapre and Deshpande
1987) or following spontaneous amplification of tandemly
repeated sequences and de novo recruitment of centromeres
and telomeres (Brachycome dichromosomatica/Asteraceae:
Houben et al. 2001; Zea mays/Poaceae: Cheng and Lin 2003).
B chromosomes are widespread in many plant and animal
groups. Unlike the corresponding m-chromosomes in
bryophytes, B chromosomes in angiosperms are of no taxo-
nomic or diagnostic significance (Guerra 2008). In
angiosperms, B chromosomes have so far been reported in
c. 1,500 species, but their distribution is non-random with
clear hot-spots (27.2% of species in Commelinales and
41.8% in Melanthiaceae in Liliales: Levin et al. 2005) and
cold-spots of occurrence (basal dicots, inbreeders: Jones
et al. 2008). Generally, monocots seem to be richer in
B chromosomes than dicots (8 vs. 3%, respectively) and
two families are particularly rich in B chromosomes:
Poaceae (152 spp.; 8.6%) and Asteraceae (171 spp.; 5.6%:
Levin et al. 2005). Although there is no notable difference
between the frequency of B chromosomes in diploids and
polyploids, there is a trend that at the family level the
frequency of B chromosomes is positively correlated with
mean 1C-genome size values (Levin et al. 2005).
B chromosomes cause intraspecific DNA content varia-
tion and may, at least partly, be responsible for reports of
chromosome number variation, especially in taxa with rela-
tively small A chromosomes and structurally variable
B chromosomes, where it is difficult to distinguish these
two types (Jones et al. 2008). B chromosomes might also
influence the evolution of the A chromosomes, for instance,
by acting as diploidizing agents for chromosome pairing in
certain allopolyploid hybrids (Evans and Macefield 1972;
Jenkins and Jones 2004) or changing recombination patterns
by affecting chiasma frequency and distribution in the A
chromosomes (Jones and Rees 1967; Jones et al. 2008).
For more details on B chromosomes see Houben et al.
(2013, this volume).
13.3.2 Chromosome Banding and Molecular
Landmarks
With the advent of molecular cytogenetic techniques, partic-
ularly in situ hybridization (fluorescence and genomic in situ
218
Fig. 13.3 Diversity of molecular chromosomal landmarks in
angiosperms. (a) DAPI C-banding in Hepatica nobilis var. japonica/
Ranunculaceae: 2n ¼ 4x ¼ 28; (b) rDNA in Melampodium
longipilum/Asteraceae: 2n ¼ 2x ¼ 20, interstitial NOR (5S rDNA in
red, 35S rDNA in green); (c) rDNA in Melampodium gracile/
Asteraceae: 2n ¼ 2x ¼ 18 (5S rDNA in red, 35S rDNA in green);
(d) Othocallis mischtschenkoana/Hyacinthaceae (formerly in genus
Scilla): 2n ¼ 2x ¼ 12 with some of 5S and 35S rDNA loci in the
same chromosome pairs (5S rDNA in red, 35S rDNA in green); (e)
GC-rich satellite DNA (Deumling 1981) in Othocallis amoena/
Hyacinthaceae (formerly in genus Scilla): 2n ¼ 2x ¼ 12; (f)
pericentric loci of satellite DNA (H Weiss-Schneeweiss et al. unpubl.)
in Prospero autumnale/Hyacinthaceae (formerly in genus Scilla): 2n
¼ 2x ¼ 12 (satellite DNA in green, 5S rDNA in red); (g) human type
telomeric loci in Othocallis siberica/Hyacinthaceae (formerly in genus
Scilla): 2n ¼ 2x ¼ 12. [All photos H. Weiss-Schneeweiss (e–g, with
D. Schweizer; f, with T.-S. Jang; both University of Vienna)]. Scale bar
represents 5 mm
hybridization [FISH and GISH, respectively]: Schwarzacher
and Heslop-Harrison 2000; Fig. 13.3), chromosomal
landmarks are no longer restricted to structural features
detectable from conventionally-stained chromosomes.
Whereas detection of some of these (NORs, telomeric
sequences) by FISH requires no de novo sequence informa-
tion (primers are conserved for larger angiosperm groups),
others (e.g., transposable elements, tandem satellite DNA
repeats) are more demanding and, therefore, their detection
has been more restricted to cultivated taxa and/or model
plants and their relatives (e.g., Nicotiana/Solanaceae: Lim
et al. 2000, 2007a; Beta/Chenopodiaceae: Dechyeva et al.
2003; Alstroemeria/Alstroemeriaceae: Kuipers et al. 2002;
Secale/Poaceae: Cuadrado and Jouve 2002). Since repetitive
DNA is the most dynamic fraction of the plant genome and
may encompass taxon-specific sequence types, it is well
suited for in depth study to characterize karyotype structure.
Being a major force in karyotype differentiation, repetitive
DNA may also indirectly contribute to race differentiation
and eventually speciation.
13.3.2.1 Heterochromatin and C-Banding
Heitz (1928) was the first to distinguish heterochromatin,
which is defined as chromosomal regions that, unlike
euchromatin, do not decondense in telophase to the same
degree as euchromatin. Heterochromatin, in contrast to
H. Weiss-Schneeweiss and G.M. Schneeweiss
diffuse euchromatin, is densely packed (and thus strongly
stained), typically replicates late in S-phase and shows
reduced rates of crossing over (Bennetzen 2000). Hetero-
chromatin usually contains only a few or no genes (e.g.,
gene-rich NOR regions) and is often enriched in repetitive
DNA (Lamb et al. 2007).
Heterochromatin rich in repetitive DNA usually remains
condensed everywhere in an organism and is thus called
constitutive heterochromatin, whereas facultative hetero-
chromatin contains less repetitive DNA and is condensed
only in some cells and/or ontogenetic stages (Brown 1966).
Recent genomic analyses have revealed that heterochroma-
tin is a term that describes many different types of condensed
chromatin, possibly with many different features and roles
and of different origin (Bennetzen 2000; Lamb et al. 2007).
Specifically, heterochromatin includes two major classes of
repetitive DNA (see Sect. 13.3.2): tandem repeats (rRNA
genes, telomeric sequences, satellite DNA) and dispersed
repeats (mostly transposable elements; Schmidt and
Heslop-Harrison 1998; Schwarzacher 2003; Gaut and
Ross-Ibarra 2008). However, the function of heterochroma-
tin remains largely unknown, even in well understood func-
tional regions such as centromeres (see also Sect. 13.3.2.6).
Euchromatin and heterochromatin distribution was first
visualized in chromosomes and interphase nuclei by classi-
cal banding techniques (mostly Giemsa C-banding: Sumner
1972; Fig. 13.3a). Such studies showed that the number and
size of bands may differ among closely related species
(Greilhuber 1982, 1995; Vosa 1985; Sumner 1990; Guerra
2000) or even within a single species, as in Scilla
(Othocallis) siberica/Hyacinthaceae (Greilhuber and Speta
1978), and in Alstroemeria aurea and A. ligtu/
Alstroemeriaceae (Buitendijk et al. 1998). Given sufficient
variation in C-band distribution, each chromosome can be
identifed (Greilhuber 1995). Despite this variability, a com-
parison of the distribution of C-bands between species has
shown that there is a general tendency for heterochromatin
to be preferentially located in subtelomeric and pericentric
regions, NORs and knobs (Bennetzen 2000; Guerra 2000). If
interstitial bands are present, these are less conserved in their
positions (Marks and Schweizer 1974; Greilhuber 1995;
Buitendijk et al. 1998). The precise position of C-bands
seems to be at least partly dependent on chromosome size,
since proximal bands prevail in karyotypes with small-sized
chromosomes (Guerra 2000).
It is noted that C-banding does not discriminate between
different types of heterochromatin but this can be accom-
plished by using fluorescent banding with base-specific
fluorochromes (Schweizer 1976, 1981). These approaches
enable most of the GC-rich and AT-rich heterochromatin
fractions to be visualized. The majority of heterochromatic
bands have been found to be rather AT-rich, and they are
more frequently found in the interstitial regions in species
13 Karyotype Diversity and Evolutionary Trends in Angiosperms
with medium and large chromosomes (Guerra 2000). 35S
rRNA genes have most often been found to coincide with
GC-rich bands (Guerra 2000). Although advanced
techniques allow detailed characterization of specific types
of repetitive DNA (see the following sections), the indis-
criminate localization of overall heterochromatin via C-
banding remains a valuable tool to enable a first and coarse
characterization of the genome, especially in poorly studied
groups.
13.3.2.2 rDNA
Genes encoding 35S (18S-5.8S-25/28S) and 5S rRNAs are
ubiquitous in eukaryotes (Richard et al. 2008). The 35S
rDNA cistron contains 18S, 5.8S and 25/28S rRNA genes
separated by the Internal Transcribed Spacers (ITS) 1 and
2 and preceded by an External Transcribed Spacer (ETS).
These cistrons are separated by the Non-Transcribed Spacer
(NTS) and are arranged tandemly (Volkov et al. 2004). 5S
rDNA repeats consist of the 121 bp 5S rRNA gene separated
by Non-Transcribed Spacers of variable length (Volkov
et al. 2004). In angiosperms, both the number and localiza-
tion of 35S rDNA and 5S rDNA loci are largely independent
from one another (Małuszyńska et al. 1998; Fig. 13.3b–d).
An exception are some clades of Asteraceae, where these
loci are physically linked (Garcia et al. 2010), and also in
some early land plants (Sone et al. 1999). The phylogenetic
distribution of such linked arrangements suggests its recur-
rent origin and/or reversal (Garcia et al. 2010). A survey of
35S rDNA loci number and distribution published for 749
species (175 genera) indicated that the average number of
35S rDNA loci per genome was 4, and that they most often
occurred at subterminal chromosomal positions (45%), usu-
ally within the short arms (Roa and Guerra 2010).
Several factors render 5S and 35S rDNA loci an excellent
choice for markers for karyotype characterization: (1) they
are ubiquitous and their coding regions are conserved over
long evolutionary distances, thus they can be localized in
various plant groups without detailed genomic information;
(2) they are highly variable with respect to localization and
number (between one and ten or even more: Małuszyńska
et al. 1998; Baeza et al. 2007) at the interspecific (Ali et al.
2005; Hasterok et al. 2006; Weiss-Schneeweiss et al. 2008),
intraspecific (Pedrosa-Harand et al. 2006) or even intrapopu-
lational level (Frello and Heslop-Harrison 2000; Baeza et al.
2007), and this variability is independent from chromosome
number; (3) they are known hotpots of intra- and
intergenomic mobility and thus might be involved in species
differentiation (Schubert and Wobus 1985; Hall and Parker
1995; Raskina et al. 2008); (4) they contain widely applica-
ble molecular markers (ITS and ETS in 35S rDNA, the non-
219
transcribed spacer of the 5S rDNA array), enabling direct
correlation of chromosomal loci evolution with phylogenetic
relationships. Although there is some redundancy with mor-
phological landmarks (morphologically discernible NORs
represent active 35S rDNA loci), the detection of 5S and
35S rDNA loci using FISH allows identification of all loci,
including small and/or inactive ones (the number of active
loci does not correlate with the overall number of loci).
Examples of successful use of one or both types of rDNA
loci in evolutionary questions are aplenty (e.g., Lim et al.
2000; Vanzela et al. 2003; Hasterok et al. 2006; Weiss-
Schneeweiss et al. 2007a, b, 2008).
The rates and mechanisms of evolutionary changes differ
between the 5S and 35S rDNA loci within the genome, and
might be group-specific. 35S rDNA loci are generally more
prone to rapid homogenization, silencing, and loss of loci,
particularly in polyploids (Clarkson et al. 2005; Kovařı́k
et al. 2005; Weiss-Schneeweiss et al. 2008; Kotseruba
et al. 2010). Both rDNA types are affected (but often to
different extents) by genome diploidization in polyploids
and this usually involves a gradual reduction of loci number,
roughly correlating with the polyploid’s age (Clarkson et al.
2005). Several mechanisms have been hypothesized to be
responsible for the high evolutionary rate and mobility,
particularly of 35S rDNA. These include, among others
unequal recombination, transposition, conversion/homoge-
nization of repeats among loci (changing the type of repeats
but not the locus position), minor loci amplification/major
loci reduction, often reversible and in hybrids usually
parent-specific loci inactivation (nucleolar dominance:
Pikaard 2000; Volkov et al. 2004) via epigenetic modi-
fications affecting the number of satellites/secondary
constrictions, and loci loss (Schubert and Wobus 1985;
Dubcovsky and Dvorak 1995; Raskina et al. 2008).
13.3.2.3 Telomeric Sequences
Telomeres are nucleoprotein structures at the ends of linear
chromosomes and are vital for protecting chromosome ends
from shortening and from being recognized and processed as
DNA breaks (Blackburn 2001; Zellinger and Riha 2007).
Telomeres in most plants are maintained by telomerase and
consist of tandem arrays of telomeric repeats. The
(TTTAGGG)n repeat characterized originally in Arabidopsis
thaliana (Richards and Ausubel 1988) is found in the major-
ity of analysed plant species (Fuchs et al. 1995) and is
considered to be ancestral for plants (Fajkus et al. 2005).
Not surprisingly, exceptions do exist, most notably in a
large clade of the monocot order Asparagales. Here, chro-
mosome termini contain the vertebrate-type telomeric
repeat (TTAGGG)n maintained by telomerase (Weiss and
Scherthan 2002; Weiss-Schneeweiss et al. 2004; Fajkus
220
et al. 2005). However, neither of these canonical telomeric
repeats is found in Allium (which is within the clade of
Asparagales with vertebrate-type telomeric sequences—
Pich and Schubert 1998), and at present the nature of the
sequences present at the telomeres of Allium chromosomes
remains unknown (Sýkorová et al. 2006).
Telomeric sequences are of relatively little use as chro-
mosomal landmarks for plant chromosomes, as they are
invariably found at chromosome ends and are conserved
(Fuchs et al. 1995; Fig. 13.3g). Unlike in animals or some
gymnosperms, telomeric-like sequences in angiosperms are
rarely found at interstitial chromosomal positions being
probably rarely retained in places of chromosomal
rearrangements (Fuchs et al. 1998; Uchida et al. 2002;
Hanmoto et al. 2007). However, de novo synthesis of
telomeric repeats in places of chromosomal breakage
stabilizes resulting chromosomal fragments (“chromosome
healing”: Tsujimoto et al. 1999) and ultimately contributes
to the evolutionary success of new karyotypic variants.
13.3.2.4 Transposable Elements
Transposable elements are a major fraction of repetitive
DNA in all eukaryotes and are one of the main drivers of
genome and chromosome size differentiation (Bennetzen
et al. 2005; Hawkins et al. 2006). In angiosperms, the most
abundant type are class I transposable elements, which trans-
pose and amplify via copy and paste mechanisms using
reverse transcriptase machinery (Kumar and Bennetzen
1999). Angiosperm genomes are particularly rich in LTR
(long terminal repeat)-retroelements of the Ty1-copia and
the Ty3-gypsy type (Kumar and Bennetzen 1999; Bennetzen
et al. 2005). Generally, retrotransposons are rather uniformly
dispersed over the entire karyotype, but they may be over-
represented in some regions (centromeres: Neumann et al.
2011; sex chromosomes: Kejnovský et al. 2009) and might
be underrepresented in others (NORs: Kumar and Bennetzen
1999; Ruas et al. 2008; but see Chester et al. 2008).
Retroelements are, in addition to polyploidy, major
players in genome size variation. There is, however, no
clear trend with respect to the type of retroelements being
involved in genome size increase. Whereas Ty3-gypsy
elements dominate in, for example, Helianthus/Asteraceae
hybrids (Ungerer et al. 2006), Fritillaria/Liliaceae
(Ambrožová et al. 2010), or Fabaceae (Vicia pannonica:
Neumann et al. 2006; Pisum sativum: Macas et al. 2007),
Ty1-copia elements dominate in Musa/Musaceae (Hřibová
et al. 2010) and Prospero autumnale/Hyacinthaceae (H.
Weiss-Schneeweiss et al. unpublished). Sometimes, a single
retrotransposon family may occupy nearly 40% of the
genome (Neumann et al. 2006).
Activation of transposable elements, often in connection
with species diversification without (Vicia/Fabaceae) or with
H. Weiss-Schneeweiss and G.M. Schneeweiss
hybridization and/or polyploidy (diploid hybrids of
Helianthus/Asteraceae: Ungerer et al. 2006; allopolyploid
Gossypium/Malvaceae: Hawkins et al. 2006), usually leads
to a uniform DNA amount increase over all chromosomes
and hence does not significantly change the overall karyo-
type structure (Bennetzen et al. 2005). However, newly
amplified retroelements also have the potential to increase
the frequency of unequal and illegitimate (ectopic) recombi-
nation. This can lead to large structural genome
rearrangements and changes in chromosome number and
results in an altered karyotype structure (Kumar and
Bennetzen 1999). In addition, some families of
retroelements may be preferentially amplified in certain
genomic regions or excluded from others (Kumar and
Bennetzen 1999; Ruas et al. 2008; Karlov et al. 2010)
leading to further changes in karyotype symmetry.
13.3.2.5 Tandemly Repeated DNA (Satellite DNA)
Satellite DNA is composed of arrays of head-to-tail arranged
basic repeat units (monomers), each usually tens to hundreds
of nucleotides long, that can exist in millions of copies in the
genome (Macas et al. 2002; Plohl et al. 2008). Satellite
repeats are among the most dynamic components of eukary-
otic genomes and are, therefore, good markers for studying
karyotype evolution within closely related plants (Lim et al.
2000; Cuadrado and Jouve 2002; Navrátilová et al. 2003;
Pires et al. 2004). Because of their rapid changes in sequence
and abundance (Macas et al. 2000, 2006), individual
families of satellite DNA are often group-specific and are
maintained as highly homogenized repeat types due to con-
certed evolution (Elder and Turner 1995). Most satellite
DNA repeat types localize in the constitutive heterochroma-
tin (see Sect. 13.3.2.1; Schmidt and Heslop-Harrison 1998;
Macas et al. 2000, 2007; Fig. 13.3e–f). Specific and novel
satellite DNA types are particularly known from sex and B-
chromosomes (Sandery et al. 1990; Kejnovský et al. 2009)
or from allopolyploids, where these novel types often
replace the most abundant types inherited from either or
both parental species (Skalicka et al. 2005; Koukalova
et al. 2010).
The genomic localization, abundance and diversity of
tandem repeats varies considerably between different plant
groups (Schmidt and Heslop-Harrison 1998; Chester et al.
2010). Some genera are characterized by containing many
different types of satellite DNA families but each family is
only weakly amplified (e.g., Pisum/Fabaceae: Macas et al.
2007; Beta/Chenopodiaceae: Menzel et al. 2008). In con-
trast, in other genera only a few satellite DNA types are
present but these may be amplified moderately (Musa/
Musaceae: Hřibová et al. 2010) or massively (Fritillaria/
Liliaceae: Ambrožová et al. 2010; Scilla (Othocallis)
siberica/Hyacinthaceae: Deumling and Greilhuber 1982;
13 Karyotype Diversity and Evolutionary Trends in Angiosperms
Prospero/Hyacinthaceae: H. Weiss-Schneeweiss et al.
unpubl., Fig. 13.3f) affecting karyotype structure and sym-
metry (de la Herrán et al. 2001). Several monocot lineages
are known for their propensity to amplify and tolerate large
amounts of satellite DNA. Due to different positions of
tandem repeat blocks in homologous chromosomes they
may be heteromorphic (Liliaceae: Peruzzi et al. 2009;
Ambrožová et al. 2010; Hyacinthaceae: Greilhuber 1995;
Crocus/Iridaceae: Frello and Heslop-Harrison 2000;
Alstroemeria/Alstroemeriaceae: Kamstra et al. 1997;
Kuipers et al. 2002; see Sect. 13.3.1.6).
It has been suggested that different satellite sequences
can coexist in genomes of related species, where they form a
library of satellite DNAs (library hypothesis: Ugarković and
Plohl 2002; Plohl et al. 2008). Initially present in different
copy numbers constituting major and minor satellite DNAs,
differential amplification after species divergence as well as
gradual and differential sequence evolution of repeats
(including loss of old and/or emergence of new satellite
types) without obvious quantitative change contribute to
the diversity of satellites DNA types (Ugarković and Plohl
2002).
13.3.2.6 Centromere-Specific Tandem Repeats
and Retroelements
Typical functional centromeres are characterized by the
presence of, among other components (see Hirsch and
Jiang 2012), specific centromeric repetitive DNA consisting
of both tandem repeats and retrotransposons (Houben and
Schubert 2003; Ma et al. 2007; Neumann et al. 2011).
Centromeric repetitive DNA is not conserved among related
plant taxa (Heslop-Harrison et al. 2003; Lee et al. 2005; Koo
and Jiang 2008; Koo et al. 2010), thus specific centromeric
tandem repeats can be used as chromosomal landmarks
once they are isolated from the given taxon/taxa group
(facilitated by the availability of ChipSeq: Johnson et al.
2007). Recently, variants of centromeric repeats used as
components of a probe cocktail for fluorescence in situ
hybridization (FISH) permitted simultaneous identifica-
tion of all chromosome pairs in soybean (Findley et al.
2010).
13.3.2.7 GISH and Chromosome Painting
Genomic in situ hybridization (GISH), first demonstrated in
synthetic cereal hybrids (Schwarzacher et al. 1989), allows
the identification of parental subgenomes in intraspecific
hybrids (Marasek et al. 2006; Choi et al. 2008) and
allopolyploids (Lim et al. 2007b; Chester et al. 2010) as
well as demonstrating their genome and karyotype evolution
(reviewed in Chester et al. 2010). The success of this tech-
nique depends, among others, on the age and genetic close-
ness of the parental taxa (Chester et al. 2010). The most
221
comprehensive information on chromosomal evolution
(including evolution of the repetitive DNA fraction) can
however be achieved via individual chromosome painting,
i.e., the identification of individual chromosomes using
chromosome-specific probes (Lysák et al. 2010). Whereas
this is state of the art in some animal groups (e.g., mammals:
Ferguson-Smith and Trifonov 2007), in angiosperms only
taxa with relatively small genome sizes (due to the low
amount of transposable elements, which by their ubiquity
limit the technique’s resolution) and extensive genomic
resources (BAC or other types of libraries, screened and
mapped to chromosomes) are amenable to chromosome
painting. So far, Arabidopsis and other Brassicaceae are
the only examples where whole-genome chromosome paint-
ing has been successfully applied using pooled BAC clones
representative of an ancestral karyotype (Mandáková and
Lysák 2008).
13.4 Specialized Chromosomes and
Chromosome Systems
13.4.1 Sex Chromosomes
In angiosperms, dioecy is found in 5–6% of genera
distributed in c. 75% of families (Renner and Ricklefs
1995; Vyskot and Hobza 2004). Of these, a few have devel-
oped differentiated sex chromosomes, which have evolved
from regular autosomal chromosome pairs (Vyskot and
Hobza 2004) via repression of recombination between
proto-sex chromosomes and accumulation of sex-specific
genes (for details see Janoušek et al. 2013, this volume).
Initially, sex chromosomes will be morphologically indis-
tinguishable from the autosomes (homomorphic sex chro-
mosomes; Carica papaya/Caricaceae: Liu et al. 2004).
However, in later stages of sex chromosome evolution they
become morphologically differentiated (heteromorphic sex
chromosomes; chromosome X and Y in Cannabis and
Humulus/Cannabaceae, Silene/Caryophyllaceae, or Coccinia/
Cucurbitaceae; reviewed in Vyskot and Hobza 2004) usually
by the accumulation of repetitive DNA (transposable
elements, satellite DNA) and subsequent changes in size and
structure of one or both sex chromosomes (Navajas-Pérez
et al. 2006; Jamilena et al. 2008; Kejnovský et al. 2009). Sex
determination either employs the active Y system (Silene
latifolia/Caryophyllaceae) or an X to autosome dosage sys-
tem, whereby sex is determined by the ratio of the number of X
chromosomes to the number of autosome sets (e.g., 2:2 in
females vs. 1:2 in males in Rumex acetosa/Polygonaceae and
Humulus/Cannabaceae: Vyskot and Hobza 2004; see also
Janoušek et al. 2013, this volume).
222
13.4.2 Permanent Translocation Heterozygotes
Translocation and inversion heterozygosity is quite frequent
within populations of many species (Levin 2002). A few
plant groups, however, have developed permanent heterozy-
gosity, where none of the chromosomes in the complement
has a fully homologous partner, although the sum of chro-
mosomal arms is constant. It is known in at least 57 species
(Levin 2002), mostly in Onagraceae (50 species of
Oenothera: Cleland 1972), but also Commelinaceae
(Rhoeo discolor: Golczyk et al. 2005) or Paeoniaceae
(Paeonia brownii and P. californica: Zhang and Sang
1998). It is considered to originate recurrently with all tran-
sitional forms present (Levin 2002). Heterozygosity is
maintained via the existence of two multichromosomal
superlinkage groups (Renner complexes: Cleland 1972),
and results in extreme inbreeding (homozygotes are lethal:
Stebbins 1950). Meiotic pairing is also peculiar in these
plants as the chromosomes form a ring when pairing,
followed by anaphase during which the centromeres are
distributed into the daughter cells in a strictly alternating
way. This results in the chromosomes of the original parental
sets staying together in either of the daughter cells
(Hejnowicz and Feldman 2000).
13.5 Karyotype Evolution in the Genomic Era
Comparative genetic and physical maps have enabled
detailed insights into the structure and evolution of the
coding part of the genome, as well as into rates and timing
of gross chromosomal changes in several genetically well
characterized angiosperm groups (Solanaceae: Wu and
Tanksley 2010; Cucumis/Cucurbitaceae: Huang et al. 2009;
Brachypodium/Poaceae: Vogel et al. 2010). At the chromo-
somal level, mapping available bacterial artificial
chromosomes (BACs) via multicolor FISH on homoeo-
logous chromosomes of related species (e.g., cross-species
multicolor BAC–FISH) offers a unique tool for comparative
genomic analyses bridging genetic and physical maps. Addi-
tionally, it allows detailed analyses of the size, position, and
evolution of repeat-rich regions of the genome (Tang et al.
2008; Lysák et al. 2010; Szinay et al. 2010). For the majority
of plants, however, comprehensive analyses of the repetitive
fraction of the genome have remained difficult, mostly
because the development of new specific chromosomal
markers has, until now, been very laborious.
The recent introduction of next generation sequencing
(NGS) and its bioinformatic analyses has brought genomic
approaches within reach for any plant biologist, providing a
H. Weiss-Schneeweiss and G.M. Schneeweiss
rapid way to identify new repeats. These techniques are
applicable to any plant group, even if no prior genomic
information is available (although basic information on
genome size and chromosome number is clearly helpful),
and at modest costs. Although one may consider traditional
karyotype analysis obsolete because a thorough genome
characterization or even whole genome sequencing can be
achieved via NGS, it is in fact quite the opposite for two
reasons: (1) basic karyotypic information, such as chromo-
some number and size and organization, genome size, or
position of landmarks, will remain important, as it cannot
easily be retrieved from NGS data irrespective of their
amount, but is necessary for correct data interpretation; (2)
the composition of plant genomes containing large amounts
of repetitive DNA is no less an obstacle for whole genome
assembly from NGS data than it was for genome assembly
from traditional Sanger sequencing data. NGS is, thus, a
perfect approach with which to rapidly extend the available
toolbox for in-depth karyotype analyses. The main challenge
for the near future will be to overcome the NGS-inherent
bottleneck of bioinformatic data analyses, especially with
respect to plant repetitive DNA, where bioinformatic tools
are notoriously scarce (but see Novák et al. 2010).
Most importantly, due to the tremendous amount of data,
NGS allows detailed insights into plant genome composition
and dynamics of different types of repetitive DNA. Apart
from a general characterization of the repeat types, including
identification of new retroelement families (Macas et al.
2007; Swaminathan et al. 2007; Wicker et al. 2009), NGS
also enables rapid isolation and characterization of novel
tandem repeat families (Macas et al. 2007; Hřibová et al.
2010), which until now required highly laborious and not
always overwhelmingly successful approaches. Importantly,
NGS for the first time provides us with the means to conduct
comprehensive comparative analyses on the genomic repeat
composition of any plant species (Macas et al. 2007;
Hřibová et al. 2010) and their contribution to genome size
change (Tenaillon et al. 2011). It also allows one to test
broader hypotheses concerning group-specific patterns of
repetitive DNA evolution, for instance, large-scale amplifi-
cation of one/few families of tandem repeats versus small-
scale amplifications of numerous small repeat families
(Macas et al. 2007; Swaminathan et al. 2007; Ambrožová
et al. 2010; Hřibová et al. 2010; H. Weiss-Schneeweiss et al.
unpubl.), or insights into retroelement-mediated faster
genome rearrangement rates suggested for monocots (Leitch
et al. 2010).
Acknowledgments The authors acknowledge the financial support of
the Austrian Science Fund (FWF), especially projects T218 (Hertha-
Firnberg Fellowship) and P21440 to HWS.
13 Karyotype Diversity and Evolutionary Trends in Angiosperms
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 Karyotype Variation and Evolution
in Gymnosperms 14
Brian G. Murray

Contents 14.1 Introduction

14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
14.2 Survey of Chromosome Number and Size Variation 232
The extant gymnosperms are the modern representatives of
the most ancient group of seed-bearing plants that first
14.3 Incidence of Polyploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
appeared in the Carboniferous, approximately 300 million
14.4 B Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 years before the present. The gymnosperms have a world-
14.5 Sex Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 wide distribution and form the dominant component of many
temperate forests in both the Northern and Southern
14.6 Karyotype Diversity and Chromosome Banding
Patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Hemispheres. They are also widely grown in plantation
forests and provide the majority of timber for construction
14.7 In situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
14.7.1 Ribosomal DNA (rDNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 and wood pulp for paper making as well as drugs and other
14.7.2 Telomere Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 phytochemicals. They are grouped into four sub-classes,
14.7.3 Other Repeats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 Ginkgooidae (one monotypic genus Ginkgo), Gnetidae
14.8 Karyotype Homology Across Species and Genera . . . 239 (Gnetum, Ephedra and Welwitschia), Cycadidae (the
cycads) and the Pinidae (the pines and other conifers). The
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
first three of these sub-classes are relatively small with just
one species in the Ginkgooidae, approximately 50 in the
Gnetidae and about 250 in the Cycadidae; the majority of
gymnosperms, some 550 species, belong to the Pinidae.
Although most gymnosperms are substantial trees, with
the redwood of California (Sequoia sempervirens) and
kauris (Agathis) of South East Asia and Oceania as promi-
nent examples, there are some like the pigmy pine,
Lepidothamnus laxifolius, that are small shrubs that seldom
grow to more than 1 m in height and some Gnetum species
that are lianes. The evolutionary relationships and taxo-
nomic positions of these four groups of gymnosperms
remain to be fully resolved (Rai et al. 2008; Zgurski et al.
2008; Chase and Reveal 2009; Eckenwalder 2009). Some
studies have placed Gnetales either as the sister group to all
conifers (‘gnetifer’ hypothesis, Chaw et al. 2000) or within
conifers as sister to cupressophytes (¼ ‘gnecup’ hypothesis;
e.g., Chumley et al. 2008) or Pinaceae (¼ ‘gnepine’ hypoth-
esis, e.g., Werner et al. 2009). In contrast, others have found
little or no support for a sister-group relationship between the
B.G. Murray (*)
School of Biological Sciences, The University of Auckland, 3 Symonds
conifers and Gnetidae (Palmer et al. 2004). Within both
Street, Private Bag 92019, Auckland Mail Centre, Auckland 1142, the conifers and cycads there has also been disagreement
New Zealand on the number and relationships of their component families
e-mail: b.murray@auckland.ac.nz

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 231

DOI 10.1007/978-3-7091-1160-4_14, # Springer-Verlag Wien 2013
232
with, for example, the maintenance of Cephalotaxaceae and
Phyllocladaceae as separate families (Page 1990) or their
merger into Taxaceae and Podocarpaceae, respectively (Rai
et al. 2008; Eckenwalder 2009). In the cycads Page (1990)
recognizes four families whereas Chaw et al. (2005) recognize
just the Cycadaceae and Zamiaceae as separate families. In this
chapter the conifer classification of Eckenwalder (2009) and
the cycad one of Chaw et al. (2005) have been adopted and the
four groups of gymnosperms are considered as sub-classes as
proposed by Chase and Reveal (2009).
14.2 Survey of Chromosome Number
and Size Variation
The gymnosperms are probably the best studied group of land
plants with regard to chromosome number. Table 14.1 lists
the 83 genera in the four subclasses of gymnosperms and
shows that chromosome numbers are known for some species
in all but three of the genera, Austrotaxus, Neocallitropsis and
Papuacedrus, all monotypic genera found either on New
Caledonia or New Guinea.
One remarkable feature of this survey is the narrow range in
overall chromosome numbers, from 2n ¼ 14 to 66, a c. five-
fold variation; this contrasts with a range from 2n ¼ 4 to c.
640, a 160-fold variation in angiosperms (Weiss-Schneeweiss
and Schneeweiss 2013). A closer examination of individual
families, where there is more than one genus, (Table 14.1)
shows an even greater uniformity. In Cupressaceae, 26 of the
27 genera that have been counted have the same basic number
of x ¼ 11, in Pinaceae nine of the 11 genera have x ¼ 12, one,
Pseudolarix, has x ¼ 11 and the other, Pseudotsuga has basic
numbers of both x ¼ 12 and x ¼ 13. Even in Podocarpaceae,
the family with the widest range of basic numbers, these range
only from x ¼ 9 to x ¼ 14. In cycads, Cycadaceae has eight
species of Cycas with 2n ¼ 22 while Zamiaceae has a wider,
though still not great, range of numbers from 2n ¼ 16 to
2n ¼ 28. The three families of Gnetidae, Ephedraceae,
Gnetaceae and Welwitschiaceae are all monogeneric. Ephedra
species are generally 2n ¼ 14 or 2n ¼ 28 although a few
hexaploid and one octoploid cytotypes have been reported (see
Leitch and Leitch 2013), Gnetum species are 2n ¼ 22 or
2n ¼ 44 while the single species of Welwitschia has
2n ¼ 42. The single species in Ginkgooidea, Ginkgo biloba,
has 2n = 24.
In the majority of gymnosperm genera, all species have
the same basic number, the exceptions include Halocarpus,
Lepidothamnus, Nageia, Prumnopitys and Podocarpus in
the Podocarpaceae, Fokenia in Cupressaceae and Zamia in
Zamiaceae. Amentotaxus argotaenia in Taxaceae is the only
species in the Pinidae to show possible intraspecific varia-
tion in chromosome number, Chuang and Hu (1963)
reported a count of 2n ¼ 14, Guan et al. (1993) reported
B.G. Murray
2n ¼ 40 and Zhou et al. (2000) found 2n ¼ 36. All three
papers have clear photographic illustrations of the
chromosomes so this variation is not easily explained.
Zhou et al. (2000) suggested that the material examined by
Chuang and Hu was A. formosana, however, Eckenwalder
(2009) does not recognise this as a distinct species. Zhou
et al. (2000) point to a similar number of chromosome arms,
the nombre fondamental (NF; Matthey 1945), to explain the
difference in their count from that of Guan et al. (1993). It
would appear that more work is needed on the chromosomes
of this species or species complex. In the Cycadidae, Zamia
loddigesii provides a striking example of chromosome num-
ber and karyotype variation, with five different chromosome
numbers and varying numbers of metacentrics and
telocentrics in populations from the Yucatan peninsula
(Vovides and Olivares 1996). This contrasts with the unifor-
mity within a species found in other cycad genera.
Gymnosperm chromosomes are characteristically large
(Fig. 14.1). A sample of measurements from three genera
shows that the maximum and minimum metaphase chromo-
some lengths within each genus were 6.4–16.2 mm for 36
species of Pinus, 5.4–14.5 mm for 16 species of Picea and
4.4–11.6 mm for six species of Larix (Hizume 1988). A
survey of genome size (C-value) variation (Murray 1998)
found a relatively small range of values, 14.4-fold compared
to nearly 2,400-fold in angiosperms (Pellicer et al. 2010;
Leitch and Leitch 2013, this volume) and, with the exception
of Gnetum, all species in that survey had 2C DNA amounts
greater than 13 pg. Since then the amount of data has nearly
doubled, with values now available for 204 species in the
Gymnosperm DNA C-values database (Leitch et al. 2001;
Murray et al. 2010). Yet despite this additional data, genome
sizes still only range 16-fold from 2C ¼ 5.572.0 pg. These
relatively large C-values together with the low chromosome
numbers typical of most gymnosperms explain the large size
of individual chromosomes in gymnosperm karyotypes.
14.3 Incidence of Polyploidy
Polyploidy is exceedingly rare in most gymnosperm
subclasses and this has been commented upon by many
authors over the years (Khoshoo 1959; Williams 2009;
Fawcett et al. 2013, this volume). It is most widespread in
Cupressaceae, with examples in Cryptomeria, Fitzroya,
Juniperus, Sequoia and Taiwania. Fitzroya and Sequoia
are both monotypic with 2n ¼ 4x ¼ 44 and 2n ¼ 6x ¼ 66
respectively, high numbers for gymnosperms. In contrast,
Cryptomeria, Juniperus and Taiwania contain both
diploid and polyploid species. Pseudolarix in Pinaceae,
with 2n ¼ 44, is also probably a derived polyploid as
all other species in the remaining 10 genera of the family
have 2n ¼ 24. The chromosome numbers reported for
14 Karyotype Variation and Evolution in Gymnosperms 233

Table 14.1 Summary of chromosome numbers in gymnosperms. – ¼ genera where no data are available
Subclass Family Genus 2n (No. of species counted)
Ginkgoidae Ginkgoaceae Ginkgo 24 (1)
Pinidae Araucariaceae Agathis 26 (2)
Araucaria 26 (13)
Wollemia 26 (1)
Cupressaceae Actinostrobus 22 (1)
Athrotaxis 22 (3)
Austrocedrus 22 (1)
Callitris 22 (11)
Calocedrus 22 (6)
Chamaecyparis 22 (6)
Cryptomeria 22, 33, 44 (2)
Cunninghamia 22 (3)
Cupressus 22 (12) 2 with Bs
Diselma 22 (1)
Fitzroya 44 (1)
Fokienia 22, 24
Glyptostrobus 22 (1)
Juniperus 22 (15) 44 (2)
Libocedrus 22 (3)
Macrobiota 22 (2)
Metasequoia 22 (1)
Microbiota 22 (1)
Neocallitropsis –
Papuacedrus –
Platycladus 22 (1)
Sequoia 66 (1) 1 with Bs
Sequoiadendron 22 (1)
Taiwania 22, 33 (2)
Taxodium 22 (3) 1 with Bs
Tetraclinis 22 (1)
Thuja 22 (4)
Thujopsis 22 (1)
Widdringtonia 22 (2)
Podocarpaceae Acmopyle 20 (1)
Afrocarpus 24 (1)
Dacrycarpus 20 (3)
Dacrydium 20 (6)
Falcatifolium 20 (1)
Halocarpus 18 (1) 22 (1) 24 (1)
Lagarostrobos 30 (1)
Lepidothamnus 28 (1) 30 (2)
Manoao 20 (1)
Microcachrys 30 (1)
Microstrobos 26 (2)
Nageia 20 (1) 26 (1)
Parasitaxus 36 (1)
Phyllocladus 18 (3)
Prumnopitys 36 (1) 38 (1)
Podocarpus 34 (2) 38 (2)
Retrophyllum 20 (3)
Saxegothaea 24 (1)
Sciadopityaceae Sciadopitys 20 (1)
Taxaceae Austrotaxus –
(continued)
234 B.G. Murray

Table 14.1 (continued)

Subclass Family Genus 2n (No. of species counted)
Amentotaxus 14, 36, 40 (2)
Cephalotaxus 24 (8)
Pseudotaxus 24 (1)
Taxus 24 (8) 1 with Bs
Torreya 22 (3)
Pinaceae Abies 24 (23)
Cathaya 24 (1)
Cedrus 24 (2)
Keteleeria 24 (2)
Larix 24 (18)
Nothotsuga 24 (1)
Picea 24 (28) 6 with Bs
Pinus 24 (68) 1 with Bs
Pseudolarix 44 (2)
Pseudotsuga 24 (3), 26 (1)
Tsuga 24 (8)
Cycadidae Cycadaceae Cycas 22 (12)
Zamiaceae Bowenia 18 (2)
Ceratozamia 16 (6)
Chigua 18 (1)
Dioon 18 (3)
Encephalartos 18 (8)
Lepidozamia 18 (2)
Macrozamia 18 (9)
Microcycas 26 (1)
Stangeria 16 (1)
Zamia 16–28 (35)
Gnetidae Ephedraceae Ephedra 14 (10) 28 (9) 14 & 28 (2)
56 (2), 1 with Bs
Gnetaceae Gnetum 22 (1) 44 (1)
Welwitschiaceae Welwitschia 42 (1)

Fig. 14.1 (a) Mitotic chromosomes of Manoao colensoi showing a of Pinus radiata; (c) CMA-banded chromosomes of Pinus radiata; (d)
‘typical’ gymnosperm complement comprising a low number of large DAPI-banded chromosomes of Pinus radiata. Scale bar ¼ 10 mm
metacentric and acrocentric chromosomes; (b) C-banded chromosomes
14 Karyotype Variation and Evolution in Gymnosperms
Amentotaxus (Taxaceae) are 2n ¼ 14, 36 and 40 (Chuang
and Hu 1963; Guan et al. 1993; Zhou et al. 2000) which is
suggestive of polyploidy in one of the species. In Gnetidae
there is one reported case of polyploidy in Gnetum while in
Ephedra it appears more common with several species com-
prising both diploid and polyploid subspecies (Table 14.1).
Welwitschia with 2n ¼ 42 would also appear to have
undergone polyploidy in its ancestry (Khoshoo and Ahuja
1963) although available molecular data is equivocal (Cui
et al. 2006).
14.4 B Chromosomes
Only 13 species spread across seven genera have been
reported to have B chromosomes (Table 14.1). These are
typically large, approximately 1/3–1/4 the length of the
largest chromosome of the normal complement (Teoh and
Rees 1977; Hizume et al. 1989, 1991). The relative rarity of
B chromosomes in gymnosperms is interesting, given that
Trivers et al. (2004) reported a positive correlation between
the presence of B chromosomes and increasing genome size
in angiosperms. Consequently, we might then expect
gymnosperms, with characteristically large genome sizes,
to have a large number of species with Bs. It is possible
that B chromosomes have a more widespread systematic
distribution because in many instances only a few indi-
viduals per species have been analysed. A more detailed
discussion of B chromosomes in plant genomes is given by
Houben et al. (2013, this volume).
14.5 Sex Chromosomes
Although the majority of gymnosperm species are hermaph-
rodite and monoecious, many are dioecious including
Ginkgo biloba, all Cycadidae and Gnetidae as well as spe-
cies scattered in many families of the Pinidae (Givnish
1980). In Ginkgo, claims have been made that the number
of chromosomes with satellites (¼ secondary constrictions)
differed between males and females and some of these
findings have been summarized by Hizume (1997). How-
ever, he analysed 10 male and 15 female plants and found
that while the number of satellites did vary, this variation
was not related to the sex of the plant. Similar claims have
been made about sex chromosomes in cycads (for example,
Sangduen et al. 2009). A heteromorphic pair associated with
sex differences was described in Stangeria by Marchant
(1968) while a clear difference in one pair of chromosomes
of Cycas revoluta (homomorphic in females and heteromor-
phic in males) was described by Segawa et al. (1971). In both
examples the male is the heterogametic sex. However, some
doubt surrounds these observations as subsequent studies in
235
Cycas revoluta have not reported a heteromorphic pair in
males (Hizume et al. 1992b; Hizume 1995), while in several
Zamia species where both male and female plants have been
analysed, no obvious chromosome differences between the
sexes have been found (Tagashira and Kondo 1999). Never-
theless, Kokubugata and Kondo (1994) have shown that
chromosome condensation in Cycas revoluta, which like
most cycads has very large chromosomes, is highly variable
and consequently the identification of homologous pairs in
the absence of differential chromosome banding and FISH
may be unreliable. More recently Hizume et al. (1998a)
applied molecular cytogenetic techniques to this problem
in C. revoluta and were able to distinguish a heteromorphic
pair, one member of which lacked a large terminal segment
of AT-rich, telomere-related DNA, in the karyotype and a
heteromorphic bivalent at metaphase I, both present only in
male plants. These results suggest an XYmale/XXfemale
sex chromosome system may be present in this species.
Other gymnosperms have different sex chromosome
systems. For example, Podocarpus macrophyllus (native to
southern Japan and China) is interesting in that it is the first
example of an XXY sex chromosome system in
gymnosperms (Hizume et al. 1988b). Males have 2n ¼ 37
and females have 2n ¼ 38, with a unique large, submetacen-
tric chromosome present in all males but absent in females.
In contrast, Davies et al. (1997) were unable to identify sex
chromosomes in any of the New Zealand Podocarpaceae,
several genera of which are dioecious. A more extensive
discussion of plant sex chromosomes is given by Janoušek
et al. (2013, this volume).
14.6 Karyotype Diversity and Chromosome
Banding Patterns
Initial chromosome studies of many gymnosperm families
using solid (Feulgen, orcein or carmine) staining suggested a
high degree of uniformity in overall karyotype structure,
However, more detailed studies, such as those of Hizume
(1988), have shown that within this apparent uniformity
there is significant, often genus-specific, variation. Hizume
(1988) classified the karyotypes of Pinaceae into seven
groups that differed in the degree of karyotype symmetry.
For example, Pinus is characterized by species having large
metacentric chromosomes with one or two pairs of smaller,
often sub-metacentric ones. In contrast, other genera such as
Larix and Pseudolarix show karyotypes with varying
degrees of bimodality.
Despite their overall structural similarity, studies using
differential chromosome banding and fluorescence in situ
hybridization (FISH) have shown that the chromosomes of
species in genera like Pinus are far from uniform (Figs. 14.1
and 14.2).
236
Fig. 14.2 In situ hybridization of repetitive DNA sequences to gym-
nosperm chromosomes. (a) 45S rDNA in Podocarpus totara; (b) 45S
and 5S rDNA (red), ‘Arabidopsis type’ telomere repeat (green) and a
centromeric repeat, PCSR, (magenta) on chromosomes of Pinus
densiflora; (c and d) colocalization of the 45S (c) and 5S (d) rDNA
hybridization sites in the F1 hybrid Podocarpus lawrencei  P. nivalis;
(e, f and g) the ‘Arabidopsis type’ telomere repeat in Cycas revoluta;
(e) Podocarpus totara (f) and Cryptomeria japonica (g) [(a, c, d and f)
from Murray et al. 2002 by permission of Oxford University Press; (b,
e and g) with permission from Dr M Hizume and the Editors of
Chromosome Science]
This was first demonstrated following studies which
showed that the Giemsa C-banding technique could be
used to locate heterochromatin in gymnosperms such as
Cycas revoluta, Ginkgo biloba and Pinus densiflora (Tanaka
and Hizume 1980). For example, MacPherson and Filion
(1981) examined five species of Pinus, two in sub-genus
Strobus and three in sub-genus Pinus, and found that
pericentromeric C-bands were limited to species in sub-
genus Pinus. Further differences between the five species
were seen in the number and position of intercalary bands. In
other Pinus species differences between C-banding patterns
have also been reported, including examples where no con-
sistent C-bands were seen (Kupila-Ahvenniemi and Hohtola
1977; Jacobs et al. 2000).
Many more species of gymnosperm have been
investigated with the base-specific fluorochromes
chromomycin A3 (CMA) and 40 ,6-diamidino-2-phenylindole
(DAPI) that stain preferentially GC or AT rich regions
respectively of heterochromatin in the chromosomes
B.G. Murray
(Fig. 14.1c, d). These staining techniques have revealed
that the amount and distribution of these two types of hetero-
chromatin can vary greatly. Only a limited number of bands
are seen in some species such as Ginkgo biloba, which shows
just four CMA-positive bands that are also C-bands (Hizume
1997). Similarly, in eight species of Taxodiaceae, Hizume
et al. (1988a) found no DAPI bands and six species had only
two or four prominent CMA bands. An absence of DAPI
bands was also reported in all the New Zealand endemic
gymnosperms belonging to three families, Araucariaceae,
Cupressaceae and Podocarpaceae, and each species had just
a single, prominent CMA-positive band (Davies et al. 1997).
In contrast, two other Cupressaceae species Cunninghamia
lanceolata and Metasequoia glyptostroboides showed small
bands at all their centromeres in addition to two terminal or
six proximal large, prominent bands, respectively. Many
species of Pinus and Larix also have large numbers of either
or both CMA and DAPI bands. For example, recent studies
of Pinus heldreichii and P. taeda have demonstrated multiple
DAPI bands distributed on all chromosomes (Bogunic et al.
2006; Islam-Faridi et al. 2007). Similarly, Hizume et al.
(1993) studied six European Larix species and found they
all had numerous CMA and DAPI bands although their
number and position differed between species.
In the Cycadidae, as in Pinidae, there are contrasting
patterns of fluorescent chromosome banding between spe-
cies and genera. Kokubugata and Kondo (1996) found that
four species of Cycas with essentially similar karyotypes
showed a common pattern of DAPI and CMA bands, with
DAPI bands at the centromeres of all chromosomes and
CMA bands widely distributed on most chromosomes at
one or both telomeres. A few interstitial CMA bands were
also seen on different chromosomes of the various species.
In subsequent studies of Zamiaceae (Kokubugata and Kondo
1998; Tagashira and Kondo 1999), no DAPI bands were
observed in Microcycas calocoma but they were seen as
small dots at the centromeres of all chromosomes of
Ceratozamia mexicana and at four to 12 centromeres of
nine different species of Zamia. Only two CMA bands
were seen in a centromeric location on the single metacentric
pair of chromosomes in M. calocoma but in C. mexicana the
CMA bands were located terminally on seven chromosomes.
CMA bands in Zamia are very variable; in some species such
as Z. muricata 20 CMA bands have been observed whereas
in Z. angustifolia only six are reported (Tagashira and
Kondo 1999).
Intraspecific variation in the number and size of CMA
bands has been reported in several species of Cupressaceae
(Hizume et al. 1988a), Pinaceae (Hizume and Tanaka 1990;
Hizume and Akiyama 1992) and Podocarpaceae (Davies
et al. 1997). For example, in Pseudotsuga menziesii
(Pinaceae) and Dacrydium cupressinum (Podocarpaceae)
bands in comparable positions on homologous chromosomes
14 Karyotype Variation and Evolution in Gymnosperms
show clear size differences (Hizume and Akiyama 1992;
Davies et al. 1997) and in Picea brachytyla var. complanata
(Pinaceae) a survey of 35 seedlings showed that though all
shared some common bands others had additional unique
bands (Hizume et al. 1991).
14.7 In situ Hybridization
Fluorescence in situ hybridization (FISH) has been used
extensively to locate a variety of repetitive DNA sequences
on chromosomes of Ginkgo biloba and many species in
Pinidae and Cycadidae.
14.7.1 Ribosomal DNA (rDNA)
Both the 18S-5.8S-26S (denoted hereafter as 45S) rDNA and
5S rDNA repeats have been mapped in a huge variety of
gymnosperms from all the subclasses except Gnetidae
(Table 14.2).
In many cases this has enabled the identification of indi-
vidual chromosome pairs in the often uniform gymnosperm
karyotype. The number and location of 45S sites varies
greatly (Fig. 14.2) from a single pair in some Podocarpus
(Murray et al. 2002), cycad (Kokubugata et al. 2000) and
Larix (Hizume et al. 1995) species to a massive 38 sites in
Pinus taeda (Islam-Faridi et al. 2007). The size of the
hybridization sites also varies greatly both between
chromosomes within a species and also between species.
Many are large and are usually at an interstitial position,
though in some cycads and Podocarpus species they are
adjacent to the telomeres (Kokubugata and Kondo 1998;
Kokubugata et al. 2000; Murray et al. 2002) (Fig. 14.2).
Small, often variable sites, in addition to the major ones,
have been reported at the centromeres of some Pinus
species (Fig. 14.2) (Liu et al. 2003; Islam-Faridi et al. 2007)
and this location has been confirmed by Hizume et al.
(2001) who used microdissection followed by degenerate
oligonucleotide-primed PCR and cloning to identify these
sequences and use them as probes in FISH experiments on
the chromosomes of Pinus densiflora.
There is considerably less variation in the number of 5S
sites with two or four being the predominant number
(Table 14.2). The 5S and 45S rDNA sites often occur on
the same chromosome and Puizina et al. (2008) have
summarized the linkage patterns of these sites in several
genera of the Pinaceae. They found that Pinus shows the
greatest diversity of patterns and that all the patterns seen in
other genera, such as Picea, Abies, Pseudotsuga and Larix,
are also found in Pinus. In several species of Podocarpus,
the 45S and 5S signals colocalize to very similar, if not the
same site in an interspersed fashion on one pair of
237
chromosomes (Fig. 14.2c, d) (Murray et al. 2002). This
pattern of linkage has also been described in liverworts
(Sone et al. 1999) and angiosperms (Garcia et al. 2007).
The diversity of rDNA locations is probably a consequence
of their movement by transposition (Datson and Murray
2006) as meiotic analyses (see Sect. 14.8 below) suggest
little structural rearrangement in the evolution of gymno-
sperm genomes.
14.7.2 Telomere Sequences
The presence of Arabidopsis-type telomere sequences
(TTTAGGGn) in gymnosperms was first demonstrated
using FISH in Pinus sylvestris and Zamia furfuracea
(Fuchs et al. 1995). They found the expected hybridization
sites at the ends of all the chromosomes of both species but
in P. sylvestris large interstitial sites were also seen. Since
then other species have been investigated and a variety of
patterns has emerged. In Cycas revoluta all chromosomes
have terminal sites that are conspicuously large on the telo-
centric chromosomes of the complement. In addition there
are sites at the centromeres of the acrocentric chromosomes
(Hizume et al. 1998a; Fig. 14.2e). Large interstitial signals,
up to four on some chromosomes, have since been reported
in all Pinus species examined (Lubaretz et al. 1996; Hizume
et al. 2000; Schmidt et al. 2000; Hizume et al. 2002a; Islam-
Faridi et al. 2007). In four species of Podocarpus, Murray
et al. (2002) found terminal sites and a dispersed pattern of
small sites scattered along the length of all chromosomes
(Fig. 14.2f). Sites at the ends of telocentric chromosomes
were not conspicuously different from those at the ends
of non-telocentric chromosomes in these species. Chromo-
somes with only terminal hybridization sites have been
reported in Abies alba, Cryptomeria japonica, Ginkgo
biloba, Larix decidua and Picea abies (Fig. 14.2g) (Lubaretz
et al. 1996; Hizume et al. 2000; Puizina et al. 2008). Albeit
with a limited sample of species, it would appear as though
Pinus amongst the gymnosperms is, to date, the only genus
with exceptionally large and conspicuous interstitial telo-
mere hybridization sites (e.g., Fig. 14.2b).
14.7.3 Other Repeats
The highly repetitive nature of DNA comprising the genomes
of gymnosperms first became apparent following studies on
the reassociation kinetics of DNA from Cycas revoluta and
several species in Cupressaceae and Pinaceae (Miksche and
Hotta 1973; Rake et al. 1980; Kurdi-Haidar et al. 1983;
Kriebel 1985). A surprising finding from these studies was
how large the percentage of ‘unique’ sequences was of the
total genome, (i.e., in the region of 20–30%). Nevertheless
238 B.G. Murray

Table 14.2 The total number of 45S and 5S rDNA hybridization sites detected by FISH in different gymnosperm genomes
Species 45S 5S Reference
Abies alba 10 4 Shibata et al. (2004), Puizina et al.(2008)
Abies nordmania 10 4 Shibata et al. (2004)
Abies pinsapo 10 4 Shibata et al. (2004)
Abies sachalinensis 8 4 Shibata et al. (2004)
Bowenia serrulata 4 – Kokubugata et al. (2000)
Bowenia spectabilis 2 – Kokubugata et al. (2000)
Bowenia sp. ‘Tinaroo’ 3 – Kokubugata et al. (2000)
Ceratozamia hildae – 2 Kokubugata et al. (2004)
Ceratozamia kuesteriana – 2 Kokubugata et al. (2004)
Ceratozamia mexicana 7/8 2 Kokubugata and Kondo (1998), Tagashira and Kondo (2001), Kokubugata et al. (2004)
Ceratozamia norstogii – 2 Kokubugata et al. (2004)
Cryptomeria japonica 2, 4 – Hizume et al. (1998b)
Cycas revoluta 16 2 Hizume et al. (1992b), Hizume (1995)
Ginkgo biloba 4 – Hizume (1997)
Larix decidua 6 2 Lubaretz et al. (1996)
Larix potaninii var. macrocarpa 2 2 Hizume et al. (1995)
Microcycas calocoma 2 – Kokubugata and Kondo (1998)
Picea abies 12 2 Lubaretz et al. (1996), Siljak-Yakovlev et al. (2002)
Picea crassifolia 10a – Hizume et al. (1999)
Picea jezoenisi var. hondoensis 10 2 Hizume and Kuzukawa (1995)
Picea glauca 14 4 Brown et al. (1993), Brown and Carlson (1997)
Picea koraiensis 10a 4 Hizume et al. (1999)
Picea omorika 16 2 Siljak-Yakovlev et al. (2002)
Picea sitchensis 10 4 Brown and Carlson (1997)
Pinus densata 18 3 Liu et al. (2003)
Pinus densiflora 14 4 Hizume et al. (1992a, 2002a)
Pinus elliottii 16 6 Doudrick et al. (1995)
Pinus massoniana 20 2 Liu et al. (2003)
Pinus merkusii 16 4 Liu et al. (2003)
Pinus nigra 18 4 Hizume et al. (2002a)
Pinus radiata 20 4 Jacobs et al. (2000)
Pinus sylvestris 14 4 Lubaretz et al. (1996), Hizume et al. (2002a)
Pinus tabuliformis 24 4 Liu et al. (2003)
Pinus taeda 38 4 Jacobs et al. (2000), Islam-Faridi et al. (2007)
Pinus thunbergii 12, 14 4 Hizume et al. (1992a, 2002a)
Pinus yunanensis 20 4 Liu et al. (2003)
Podocarpus acutifolius 2 2 Murray et al. (2002)
Podocarpus lawrencei 4 2 Murray et al. (2002)
Podocarpus nivalis 2 2 Murray et al. (2002)
Podocarpus totara 2 2 Murray et al. (2002)
Pseudotsuga menziesii 6 2 Hizume et al. (1996)
Stangeria eriopus 16 2 Kokubugata et al. (2002, 2004)
Zamia angustifolia 6 2 Tagashira and Kondo (2001)
Zamia integrifolia 6 2 Tagashira and Kondo (2001)
Zamia pumila 7 2 Tagashira and Kondo (2001)
Zamia pygmaea 6 2 Tagashira and Kondo (2001)
Zamia furfuracea 14 2 Tagashira and Kondo (2001)
Zamia loddigesii 14 2 Tagashira and Kondo (2001)
Zamia skinneri 6 2 Tagashira and Kondo (2001)
Zamia vazquezii 10 2 Tagashira and Kondo (2001)
Zamia muricata 20 2 Tagashira and Kondo (2001)
a
Additional weak signals observed in some plants
14 Karyotype Variation and Evolution in Gymnosperms
the repetitive fraction still comprised a very significant
component of the genome. Elsik and Williams (2000)
suggested that the high frequency of ‘unique’ sequences
was due to the presence of divergent retrotransposons
which contributed to the excess low-copy DNA and indeed,
recent sequence data support this. In a large scale trans-
criptome sequencing project of Pinus contorta Parchman
et al. (2010) identified a surprisingly large number of
transcriptionally-active retrotransposon sequences in their
sample. While in Pinus taeda Morse et al. (2009) uncovered
large numbers of gypsy and copia retrotransposons in the low
copy fraction of a Cot-based fractionation of genomic DNA.
Further verification of Elsik and Williams’ proposal and a
clearer idea of the contribution and composition of ‘unique’
sequences in gymnosperm genomes will no doubt come as
high throughput sequencing technologies are increasingly
applied to gymnosperms.
Cafasso et al. (2003) isolated an unusual GC-rich satellite
repeat from Zamia paucijuga that was shown to be located in
the sub-terminal regions of most chromosomes using FISH.
The repeat also existed as a dispersed repeat that comprised
two to four satellite repeats flanked by a 0.6 kb AT-rich
repeat. These dispersed elements were found in 30 other
Zamia species but not in Ceratozamia and Microcycas and
the amount and dispersal pattern of the repeats differed
between Zamia species (Cafasso et al. 2009). In some spe-
cies the repeats were at low or almost undetectable levels
whereas other showed very strong signals on Southern blots
and they often showed evidence of secondary amplification
events.
In contrast to these cycad results, Brown et al. (1998)
identified an AT-rich satellite sequence from Picea glauca
that had a centromeric distribution on a sub-set of
chromosomes of P. glauca and P. sitchensis. Southern
hybridization showed that the sequence was present in 18
Picea species but absent from Pinus, Pseudotsuga and Thuja
and that amongst Picea species there was little evidence of
structural alteration of the sequence. Subsequently, Hizume
et al. (1999) used the sequence as a probe onto chromosomes
of two Chinese Picea species and found clear hybridization
signals at the centromeres of three pairs of chromosomes,
fewer than the four or five pairs found by Brown et al.
(1998). Hizume et al. (2002b) have since found a similar
distribution pattern for two AT-rich sequences, one a 170 bp
repeat and the other a 220 bp repeat that was a partial
duplication of it, both located near to the centromeres of 22
chromosomes in Larix leptolepis but only 14 chromosomes
of L. chinensis.
Various microsatellite or simple sequence repeats (SSRs)
have been identified and in some cases mapped to
chromosomes of Podocarpaceae and Pinaceae. Many
sequences represent a large fraction of the repetitive DNA in
the genome and have dispersed FISH hybridization patterns
239
showing that the sequences are widely distributed across the
chromosomes (Schmidt et al. 2000; Murray et al. 2002). In
several species, standard telomere repeats have given rise to
mutated or degenerate copies that may have non-terminal
chromosomal locations and some are present at very high
repeat frequency and constitute significant chromosome
landmarks (Schmidt et al. 2000; Shibata et al. 2005).
The distribution and evolution of retrotransposons has
also been studied in gymnosperms and, as noted above,
both gypsy and copia elements have been found (Kamm
et al. 1996; Brandes et al. 1997; Friesen et al. 2001). When
hybridized to Pinus elliottii chromosomes, a cloned Ty1-
copia-like element showed a dispersed pattern of
hybridization over all the chromosomes, with the excep-
tion of centromeres and nucleolar organizing regions
(NORs) (Kamm et al. 1996). Friesen et al. (2001) looked
at the distribution of various gypsy and copia elements in
Picea abies and Pinus pinaster. While some gypsy
elements showed stronger signals at the chromosome
ends (e.g., Pagy11 in Picea abies) or associated with 18S
rDNA and centromeric regions (e.g., Ppgy1 in Pinus
pinaster) most showed a dispersed pattern of hybridization,
similar to that reported for another gypsy element (Gymny)
in Pinus taeda (Morse et al. 2009). Indeed, such a dis-
persed distribution pattern which has been reported
for many other pine gypsy elements contrasts with
observations in angiosperms where most gypsy elements
appear localized in the centromeric and pericentromeric
regions (Grover and Wendel 2010). Such observations
hint at fundamental differences in the organization of
DNA between angiosperms and gymnosperms and this
has recently been discussed by Leitch and Leitch (2013).
14.8 Karyotype Homology Across Species
and Genera
The uniformity of Pinidae karyotypes begs the question of
whether the similarity is superficial, with overall chromo-
some numbers and morphology being conserved, or whether
variation is widespread but karyotype orthoselection (i.e.,
the maintenance of karyotype uniformity through the occur-
rence of characteristic structural mutations; White 1973)
masks extensive DNA sequence rearrangements. As
discussed above, studies using chromosome banding and
FISH have revealed extensive variation in the location of a
variety of repetitive DNA sequences, but to assess whether
chromosomal conservation at a large syntenic scale has
occurred requires evidence either from comparative linkage
analysis using orthologous loci or the analysis of chromo-
some pairing at meiotic prophase I/metaphase I in interspe-
cific hybrids. Available data from both approaches support
240
the general idea of karyotype conservation across specific
and generic divides. For example, fertile hybrids between
the Japanese Larix kaempferi (syn. L. leptolepis) and L.
decidua, from Northern and Central Europe, showed regular
meiotic pairing with a similar chiasmata and univalent fre-
quency to the parental species (Sax 1932). L. kaempferi has
also been crossed successfully with L. sibirica and L.
gmelenii (Eckenwalder 2009). Many more hybrid
combinations have been studied in Pinus (Sax 1960; Saylor
and Smith 1966; Williams et al. 2002) and these also show
that there have been few major structural changes in the
evolution of Pinus karyotypes. In Podocarpaceae, introgres-
sive hybridization has been reported between Podocarpus
totara and P. acutifolius. This implies that the hybrids are
fertile with presumably regular meiosis and hence karyotype
uniformity (Wardle 1972). Quinn and Rattenbury (1972)
studied F1 hybrids between Dacrydium laxifolium and D.
intermedium and observed mostly bivalent formation with
two to four univalents in 75% of cells and one or two
dicentric bridges and associated fragments in a similar per-
centage of anaphase I cells. They concluded that these two
species were basically similar in karyotype structure but
differentiated by two paracentric inversions. None of these
gymnosperm hybrids showed any evidence of chromosome
translocations.
Genetic maps have been produced for several species of
Pinus and these show a remarkable conservation of gene
distribution and order between species. These data have
enabled comparative analyses to be made between various
Pinus genomes and that of Pseudotsuga menziesii (Krutovsky
et al. 2004). Pseudotsuga menziesii differs from all other
Pinaceae in having 26 as opposed to 24 chromosomes, it has
11 pairs of metacentrics/sub-metacentrics and two pairs of
telocentrics compared with 12 pairs of metacentrics/sub-
metacentrics found in all other species. Comparative mapping
has shown that overall there is conservation of syntenic loci
between the species of Pinus analysed as well as between P.
taeda and P. menziesi, thus providing further support for the
proposal that Pinaceae genomes have been remarkably stable
over c. 45 million years, the time when Pseudotsuga first
appeared in the fossil record. It is possible that there has
been even greater stability as fossils corresponding to the
subsections of Pinus, which share common karyotypes, have
been found in Cretaceous floras over 65 million years old
(Stockey and Nishida 1986; Miller 1993).
The Cycadidae provide contrasting patterns of karyotype
stability in different genera. Lepidozamia, Macrozamia,
Encephalartos and Dioon, for example, all have the same
chromosome number, 2n ¼ 18, and essentially similar
karyotypes, though Encephalartos lacks a pair of telocentric
chromosomes in its complement which are characteristic of
the other three genera (Marchant 1968; Kokubugata et al.
1999; Sangduen et al. 2009). Cycas species also show essen-
tially common patterns of fluorochrome banding
B.G. Murray
(Kokubugata and Kondo 1996). In contrast, Zamia is chro-
mosomally variable with numbers ranging from 2n ¼ 16 to
2n ¼ 28. Much of this variation has been attributed to cen-
tric fission or fusion as the species with lower numbers of
chromosomes have more metacentrics and fewer
telocentrics compared with species with higher chromosome
numbers which show the reverse pattern. However the direc-
tion of change remains controversial (Marchant 1968;
Moretti 1990; Caputo et al. 1996). Whether there is large-
scale conservation of gene order in cycad chromosomes is
not known as no large-scale genomic mapping exercises
have been carried out.
Conclusions
The overall uniformity of chromosome number and kar-
yotype in the major groups of gymnosperms is in
striking contrast to the situation in angiosperms
(Weiss-Schneeweiss and Schneeweiss 2013, this vol-
ume). The observation of conserved biological form
and function is strongly susceptible to an adaptive inter-
pretation but an obvious selective explanation is
lacking for gymnosperm karyotypes. White (1973)
differentiated between karyotype orthoselection, the
maintenance of karyotype uniformity through the occur-
rence of characteristic structural mutations, and karyo-
type conservation, the maintenance of similar karyotype
morphology in different taxa through a lack of structural
mutations. Karyotype orthoselection has been put for-
ward as an explanation (Williams 2009), however it
would appear that it is karyotype conservation that is
important here as there is little evidence for widespread
structural change in most gymnosperms (with the excep-
tion of some cycads). What the selective mechanism
underlying gymnosperm karyotype conservation is
remains a significant goal for understanding the evolu-
tion of gymnosperm karyotypes.
Nevertheless, the application of molecular cytogenetic
techniques to gymnosperm genomes has revealed consid-
erable variation in the location and composition of a wide
variety of repetitive DNA sequences and this variation
has proved to be invaluable for karyotyping and chromo-
some identification. This is amply illustrated by two
examples from Pinus. In P. densiflora Hizume et al.
(2002a) and Shibata et al. (2005) used FISH to map a
variety of different repeats to the chromosomes
(Fig. 14.2b) and this has allowed the identification of all
members of the complement. Similarly, Islam-Faridi
et al. (2007) have produced a reference karyotype for P.
taeda and provided a key for the identification of each
member of the complement. Although both these studies
have used many comparable sequences Islam-Faridi et al.
(2007) point out that it is still very difficult to identify
homologous chromosomes between P. taeda and P.
densiflora. The development of a wider range of probes
14 Karyotype Variation and Evolution in Gymnosperms
and their application to a range of species should better
facilitate the comparative physical mapping of gymno-
sperm chromosomes. Mapping studies suggest the con-
servation of syntenic groups of genes (though P.
densiflora has not been included in such studies) across
many millions of years (Krutovsky et al. 2004) but
repeats such as the rDNA sequences do not appear to be
under the same constraints.
The increasing speed and falling costs of high-
throughput genome sequencing approaches are expected
to ultimately lead to the complete sequencing of genomes
such as those of Pinus and Picea, despite their large size.
Nevertheless, already transcriptome sequencing data are
available for some gymnosperm species (e.g., Pinus
contorta—Parchman et al. 2010, Ginkgo biloba—
Brenner et al. 2005) and these are providing a wealth of
novel data on the functioning and evolution of gymno-
sperm genomes (Barker et al. 2010). Such data together
with improved understanding of the phylogenetic
relationships between different gymnosperm lineages
will provide enhanced insights into the evolutionary pro-
cesses which have shaped the karyotypes of these eco-
nomically very important plants.
Acknowledgments I would like to thank Dr Masahiro Hizume for
allowing me to use some of his images to illustrate this chapter.
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 Karyotype and Genome Evolution
in Pteridophytes 15
Michael S. Barker

Contents 15.1 Introduction

15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Although genomics is a field often discussed as a recent
15.2 High Chromosome Numbers and Hypotheses . . . . . . . . . . 246
development brought upon by the advent of next-generation
15.3 Recent Advances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 sequencing technologies, the roots of genomics extend to at
15.4 Future Directions for Fern Nuclear Genomics . . . . . . . . . 251 least the early twentieth century. Rather than running a gel or
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
assembling sequence reads on a server, early cytologists
squashed and stained actively dividing cells and viewed
them under a microscope to reveal a variety of chromosome
features including numbers, sizes, and pairing behaviour.
These data revolutionized our perspective of plant species,
and provoked numerous questions about genome evolution,
some of which endure today. Among these long-standing
questions is how the high chromosome numbers of homo-
sporous ferns and lycophytes evolved and are maintained.
By the 1950s, it was clear that fern nuclear genomes,
particularly those of homosporous species, possessed excep-
tionally high chromosome numbers (Manton 1950). Com-
parison of chromosome counts among related species of
many genera clearly showed that series of currently recog-
nizable polyploids (neopolyploids) such as tetraploids and
hexaploids are frequent among ferns and lycophytes, even-
tually reaching the highest known chromosome count among
extant eukaryotes in Ophioglossum reticulatum (n > 600;
Khandelwal 1990). These observations indicated that whole
genome duplication is an ongoing process and a significant
feature of fern and lycophyte evolution. In addition to the
abundant occurrence of neopolyploids, it became clear that,
with rare exceptions, even the lowest chromosome numbers
in each genus were much higher than those of other plant
groups. Cytologists studying angiosperms and other
heterosporous species rarely required more than fingers
and toes to count chromosomes; flowering plants have an
average of n ¼ 15.99 chromosomes (Klekowski and Baker
1966). However, homosporous ferns and lycophytes
required more digits and patience to count their
M.S. Barker (*)
Department of Ecology & Evolutionary Biology, University of
chromosomes. On average, homosporous fern and lycophyte
Arizona, Tucson, USA genomes contain n ¼ 57.05 chromosomes, over three-fold
e-mail: msbarker@email.arizona.edu

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 245

DOI 10.1007/978-3-7091-1160-4_15, # Springer-Verlag Wien 2013
246
more than the average flowering plant (Klekowski and Baker
1966). This striking difference between homosporous and
heterosporous plants spawned a number of hypotheses.
15.2 High Chromosome Numbers
and Hypotheses
Although Manton’s (1950) cytological studies focused atten-
tion on homosporous plant chromosome numbers,
Klekowski and Baker (1966) provided the first synthetic
hypothesis for their origin and maintenance. Historically,
haploid chromosome numbers higher than 14 were generally
considered to be polyploids in angiosperms (Grant 1981). If
this rule were applied to homosporous ferns, with their
average n ¼ 57.05, more than 95% would be diagnosed as
polyploids. A significant exception to the generally high
chromosome numbers in ferns are the heterosporous water
ferns; the haploid chromosome number for heterosporous
ferns averages 13.6, and 90% of these species have
chromosome numbers less than 28. A similar situation is
encountered in the lycophytes, where the homosporous
Lycopodiaceae have significantly higher chromosome num-
bers than their sister groups (Pryer et al. 2004), the
heterosporous Selaginellaceae and Isoetaceae (L€ove et al.
1977). Thus, homosporous pteridophytes have significantly
higher chromosome numbers compared to their close
heterosporous relatives and seed plants.
Based on the large number of chromosomes, Klekowski
and Baker (1966) posited that most homosporous ferns were
polyploids or species with more than two complete sets of
chromosomes in their somatic cells. They proposed that
homosporous vascular plants evolved high numbers of
chromosomes and ploidy levels to provide an extra source
of genetic variation to compensate for their putative primary
mode of reproduction, intragametophytic self-fertilization, an
extreme form of inbreeding that results in 100% homozygos-
ity in a single generation (Klekowski 1973). Under this per-
spective, homosporous ferns potentially suffer severe losses
of heterozygosity more frequently than heterosporous plants.
According to Klekowski and Baker (1966), additional non-
Mendelian genetic variation could be generated by abnormal
pairing during meiosis of different versions of chromosomes,
or homoeologs, rather than the normal homologous pairing.
Homoeologous pairing would be unaffected by this extreme
self-fertilization and release genetic variation in these puta-
tively polyploid genomes that would otherwise be “fixed.”
Putatively, heterosporous ferns should have lower chromo-
some numbers than homosporous ferns because they are
obligately outcrossing and therefore do not experience
sharp reductions in heterozygosity through intragam-
etophytic selfing. A variety of subsequent studies supported
M.S. Barker
Klekowski and Baker’s hypothesis. For example, Hickok and
Klekowski (1974) and Hickok (1978) demonstrated
homoeologous pairing in a small percentage (<1–3%) of
self-fertilized offspring of the tetraploid Ceratopteris
thalictroides. These studies demonstrated that homoeologous
pairing can occur and may operate to provide heterozygosity
in otherwise largely homozygous vascular plants.
Additional data to address the Klekowski and Baker
(1966) hypothesis came from isozymes rather than additional
cytological research. By providing direct insight into patterns
of genetic variation and inheritance, isozymes were instru-
mental for characterizing fern and lycophyte genomes. An
early isozyme study allegedly supported the Klekowski and
Baker hypothesis by finding non-Mendelian inheritance of
multiple enzyme bands, consistent with homoeologous
pairing in a neopolyploid (Chapman et al. 1979). However,
a subsequent investigation demonstrated that a homosporous
fern with the lowest chromosome number for its genus pos-
sessed a diploid, not polyploid, gene expression profile with
Mendelian inheritance (Gastony and Gottlieb 1982). Further-
more, it was demonstrated that the complex isozyme banding
patterns of Chapman et al. (1979) were mostly attributable to
subcellularly compartmentalized isozymes (Gastony and
Darrow 1983; Wolf et al. 1987). Continued isozyme
investigations supported Gastony and Gottlieb’s observation
that homosporous ferns with base chromosome numbers for
their genus had diploid expression profiles despite possessing
relatively high chromosome numbers (Gastony and Gottlieb
1985; Haufler and Soltis 1986; Soltis 1986; Haufler 1987).
These results differed from those in angiosperms, which
showed that angiosperms with high chromosome numbers
did indeed have duplicated sets of isozymes (reviewed in
Gottlieb 1982; Soltis and Soltis 1990).
Analyses of fern breeding systems also cast serious doubt
on the Klekowski and Baker (1966) hypothesis. Rather than
finding evidence of extensive intragametophytic self-
fertilization, analyses of natural fern populations also
found that they were predominantly outcrossing. Haufler
and Soltis (1984) found that 83% of the examined
populations of diploid Bommeria hispida had heterozygous
enzyme banding patterns attributable to outcrossing between
genetically different gametophytes. In a more extensive
analysis of diploid populations of Pellaea andromedifolia,
Gastony and Gottlieb (1985) determined that at least 81.3%
of 145 sporophytes examined from natural populations arose
through outcrossing between gametophytes carrying differ-
ent alleles. Because of the unique breeding system of homo-
sporous vascular plants, Holsinger (1987) developed a
specialized statistical technique for estimating rates of self-
fertilization and applied it to the data of Gastony and
Gottlieb (1985). He found no evidence of intragametophytic
self-fertilization in two of Gastony and Gottlieb’s
15 Karyotype and Genome Evolution in Pteridophytes
populations with sufficient sample sizes for his statistical
analyses and concluded that intragametophytic self-
fertilization occurs only a small fraction of the time, if at
all. Thus Klekowski and Baker’s (1966) original rationale
for the polyploid homoeologous heterozygosity hypothe-
sis—high levels of intragametophytic self-fertilization—
was rejected.
By the end of the isozyme era, it was clear that the
previous explanation for high numbers of chromosomes in
homosporous genomes—high levels of neopolyploidy—was
no longer supported. Alternative explanations that do not
involve polyploidy for the high chromosome numbers,
including ancestral high chromosome numbers and high
rates of chromosomal fission, have never gained much trac-
tion because counts within genera and clades often follow a
euploid pattern and rates of neopolyploidy are high among
ferns (Wood et al. 2009). To explain the paradox of high
chromosome numbers and diploid isozyme expressions in
homosporous ferns, Haufler (1987) hypothesized that homo-
sporous ferns may have acquired high chromosome numbers
with diploid gene expression through repeated cycles of
polyploidization and subsequent gene silencing without the
loss of chromosomes. If such a mechanism is common, the
genomes of extant homosporous ferns should contain multi-
ple, silenced copies of each nuclear gene. In support of
Haufler’s hypothesis, a few studies have identified silenced
nuclear genes in homosporous fern genomes. Gastony (1991)
captured the process of gene silencing by documenting that the
duplicated expression of a phosphoglucoisomerase isozyme in
the neotetraploid homosporous fern Pellaea rufa progressively
diminished to a diploid level in several populations. However,
isozyme analyses can assess only the expression of enzyme-
coding loci and not the presence of these genes. Therefore, it
cannot be determined whether homosporous ferns with the
lowest chromosome numbers in their genus have diploid
expression of isozyme loci because they have never experi-
enced polyploidization or because they have gone through the
cycle of polyploidization and gene silencing, unless sequences
of gene copies are examined (reviewed in Soltis and Soltis
1987). Using DNA sequence based probes for chlorophyll a/b
binding protein (CAB) genes in the homosporous fern
Polystichum munitum, Pichersky et al. (1990) identified sev-
eral copies of this high copy number gene and found that
several gene copies had been pseudogenized, consistent with
Haufler’s hypothesis.
Subsequent studies evaluating gene copy number in
homosporous ferns utilized Restriction Fragment Length
Polymorphisms (RFLPs). RFLPs may be used to estimate
the number of genes present in a genome by using DNA
probes to detect length differences in restriction enzyme-
digested genomic fragments. Mutations at paralogous loci
will introduce changes in the length of fragments by either
directly introducing length differences (e.g., indels) or by
247
altering restriction sites. Using RFLPs with several cDNA
probes, McGrath et al. (1994) found multiple copies of most
genes tested in the genome of the diploid homosporous fern
species Ceratopteris richardii, consistent with Haufler’s
hypothesis. Therefore, even diploid species of homosporous
ferns seem to have multiple gene copies, although many
appear to be silenced pseudogenes. McGrath et al. also
compared the RFLP profiles of diploid C. richardii and
tetraploid C. thalictroides and expected that twice as many
gene copies should be present in C. thalictroides. In contrast
to their expectation, the tetraploid species had on average
only 30% (marginally significant) more gene copies and
some loci were single-copy. Although some paralogs may
have been undetected if they co-migrate on the gel or if their
sequences are too diverged from the probe sequences, it
seems reasonable to assume that at least in some cases,
genes duplicated through polyploidization have been
not only silenced but also completely deleted from the
genome. These results are consistent with the spectrum
of observations in angiosperm genomes and polyploids
(Wendel 2000).
15.3 Recent Advances
In the last decade, the study of plant genomes has advanced
with the advent of new sequencing technologies. Fern and
lycophyte genomics are beginning to benefit from these
advancements and progress is being made on the long-
standing questions surrounding the evolution of high chromo-
some numbers (Barker and Wolf 2010). Traditional marker
based approaches to the study of genomics often have limited
power, especially if mapping populations are not employed to
evaluate inheritance patterns. For example, RFLP studies
cannot distinguish genome-wide polyploidization events
from small scale duplications because the number of detected
RFLP fragments tends to overestimate the number of gene
copies if restriction sites exist within the probe hybridizing
sites. Critically, the relative positions of gene copies in the
genome are unknown in RFLP studies without analysis of
segregating populations. Some of these limitations can be
overcome by fluorescent in situ hybridization (FISH) or
“chromosome painting,” which allows one to visualize the
physical position of genes corresponding to the fluorescently
labelled hybridizing probes on the fixed chromosomes.
Using a ribosomal DNA (rDNA) probe, FISH analyses in
C. richardii showed that multiple rDNA gene copies exist
on different chromosomes (McGrath and Hickok 1999),
supporting the hypothesis of paleopolyploidy in homosporous
ferns. However, it is difficult to determine relative positions
among different marker loci with FISH, and the approach is
technically challenging and labour intense, especially when
hybridizing many low copy markers. Other techniques have
248
provided more information about fern and lycophyte
genomes with more reliability.
Genetic linkage mapping has provided the most compre-
hensive view of synteny, or gene order colinearity among
duplicated regions. Linkage maps describe the relative
locations of polymorphic markers in genomes inferred from
the segregation patterns of parental alleles in mapping
populations. Inference of marker positions is based on a
simple assumption: the frequency of recombination between
two markers increases with the physical distance between
these loci, although physical and genetic map distances are
often not proportional because recombination rates are
unevenly distributed across the genome. Linkage mapping
is a powerful tool for investigating paleopolyploidy not only
because gene copy number and relative marker positions can
be estimated fairly accurately, but also because mapping a
large number of duplicated genes allows us to infer large
scale duplication (e.g., polyploidy) if sets of duplicated loci
occur in similar orders in different parts of the genome.
Nakazato et al. (2006) constructed a genetic linkage
map of the homosporous fern C. richardii based on 729
markers (368 RFLPs, 358 Amplified Fragment Length
Polymorphisms (AFLPs), and 3 isozymes) using a mapping
population consisting of 488 Doubled Haploid Lines
(DHLs). Most genes (85%) were found in multiple copies
based on screens of 1,037 RFLPs hybridized to fluorescently
labelled cDNA probes, consistent with the results of previous
RFLP studies. After correcting for potential multiple restric-
tion sites within hybridizing fragments, 24% of genes were
estimated to be single copy. This is one of the lowest
proportions of single copy genes among plant species, at
least as measured by RFLP linkage mapping. Local
duplications such as tandem duplications are common, yet
a large number of duplications were detected between rather
than within chromosomes. Although these data favor the
paleopolyploidy hypothesis as in previous studies, Nakazato
et al. (2006) were unable to find conclusive evidence of
paleopolyploidy. First, the detected gene duplicates appear
to be scattered more or less haphazardly across the genome
rather than occurring in recognizable homoeologous chro-
mosomal segments (Fig. 15.1). If there was an ancient
polyploidization, syntenic chromosomal blocks would be
expected to have been recognizable even after millions of
years. For example, the genomes of Brassica species
currently recognized as diploid are mainly composed of
three sets of gene copies with short syntenic regions,
suggesting that they are ancient hexaploids (Lagercrantz
and Lydiate 1996).
The absence of significant homoeologous synteny in
Nakazato et al.’s mapping study suggests that polyploi-
dization has not occurred in at least the last several million
years in the C. richardii lineage and perhaps not in diploid
homosporous ferns in general. Statistical analyses of the
M.S. Barker
distribution of gene copies, however, indicate that there is
significant clustering in the C. richardii genome; that is, the
copies of a set of markers in a given linkage group tend to
co-occur on a different linkage group, although the pattern is
not strong enough to be visually apparent. This result
suggests that there may have been ancient whole genome
duplications in the history of C. richardii, and likely other
related polypod ferns, that have been masked by subsequent
chromosomal and gene copy number evolution.
To gain further insight into the origin of the high
chromosome numbers of homosporous ferns, modern geno-
mic tools have recently been applied to the question. In large
and putatively complex genomes such as those of ferns,
whole genome sequencing has not yet been economically
feasible. An alternative to sequencing a whole nuclear
genome is to sequence a large portion of the transcriptome,
i.e., that part of the genome that is transcribed into RNA.
Such whole-scale sequencing of cDNA libraries—either
normalized Expressed Sequence Tags (ESTs) where highly
expressed transcripts are reduced in abundance or non-
normalized RNA-Seq approaches—has been particularly
useful in ferns. Using traditional Sanger long reads or
many more but shorter next-generation technologies such
as Illumina or 454 sequencing, entire transcriptomes and
the “gene catalog” may be economically sequenced. Compu-
tational tools specifically developed for large scale sequence
analyses (e.g., EvoPipes.net; Barker et al. 2010) are used to
efficiently analyse and summarize these large data sets.
Transcriptome approaches are particularly well suited for
studying the genomics of non-model organisms because
genome complexity is reduced by focusing on transcribed
regions of the genome. Considering the low cost and analytic
flexibility of ESTs, they will likely play a significant role in
furthering our understanding of fern genomes.
How can transcriptome data be used to evaluate the
paleopolyploid hypothesis and the origin of high chromo-
some numbers in ferns? Phylogenies of each gene family
may be constructed from a transcriptome library for a spe-
cies, and common gene duplication events identified. By
plotting the ages of all gene duplications, typically in terms
of their number of synonymous substitutions (Ks or dS),
ancient genome duplications may be inferred as peaks in
the age distribution (Fig. 15.2). These peaks reflect the very
large number of duplications of similar age that one expects
to result from ancient polyploidy, against a backdrop of
ongoing small scale gene duplication birth and death. By
using relative rate corrections and multiple species gene
family phylogenies to account for variation in substitution
rates across lineages, it is possible to discern whether
paleopolyploidizations observed in two or more taxa are
shared (Barker et al. 2008, 2009). Applied to the
transcriptomes of two polypod ferns (Ceratopteris richardii
and Adiantum capillus-veneris) similar analyses have
15 Karyotype and Genome Evolution in Pteridophytes 249

Fig. 15.1 Plot of duplicated gene copies shared by pairs of linkage to the cM map length of the corresponding linkage group (After
groups for the homosporous fern Ceratopteris richardii (indicated by Nakazato et al. 2006)
the numbers). The width and the height of each block are proportional
250
Fig. 15.2 Histogram of 70
Ceratopteris richardii duplicate
Ks values demonstrating a peak in
duplications from Ks 0.96 to 1.84 60
(Barker 2009). The solid line is a
significant SiZer (Chaudhuri and
Marron 1999) kernel density
estimate, with the peak significant 50
at p < 0.05
% Duplicate Pairs
40
30
20
10
0 0
0.5
revealed each lineage shares only a single detectable
genome duplication with a median peak of Ks ~ 1.6 (Barker
2009; Fig. 15.2). By combining fossil dates (Schneider et al.
2004) with nuclear gene phylogenies from the transcriptome
data, this duplication event is thought to have occurred nearly
180 million years ago. This places the paleopolyploidization
somewhere along the branch leading to all or most of the
polypod ferns, the largest extant clade of homosporous ferns.
Although we have evidence for one ancient genome dupli-
cation in the ancestry of most extant ferns, it is unlikely that a
single paleopolyploidy over the last 200 million years is
sufficient to create and maintain the extraordinary chromosome
numbers of homosporous ferns. Differences in the rate of
chromosomal change and loss may also play a significant
role. Following multiplication, the chromosomes of a polyploid
genome may form complexes of three or more chromosomes
(multivalents) during meiosis. A distinguishing trait of diploidy
is the formation of pairs (di-ploidy) of chromosomes (bivalents)
during meioses. Although many plants have experienced at
least one round of ancient genome duplication, these species
all behave as genetic diploids rather than polyploids. So how do
most plant genomes, which have experienced rounds of ancient
genome duplications, return to this diploid genetic state?
Subsequent to genome duplication, plant nuclear genomes
undergo a series of changes that restore the diploid genetic
system, a process known as diploidization. Typical plant
genomes experience, at varying rates, rearrangements and
often a net loss of genetic material, via mechanisms such
as chromosomal fusion, illegitimate recombination, and
M.S. Barker
1.2
1
0.8
Density
0.6
0.4
0.2
1 1.5 2 2.5 3 3.5 4 4.5 5
Ks
transposition (see Lysák and Schubert 2013, this volume).
Combined with silencing of duplicated genes via mutation or
outright loss, the actions of this suite of genomic changes lead
to diploidization (Doyle et al. 2008; see also Fawcett et al.
2013, this volume). Significant variation in the rate of these
genomic changes is well known from the numerous whole
genome sequences available for the angiosperms. At one
extreme is Arabidopsis, whose small nuclear genome contains
only five chromosomes but has experienced at least three
rounds of whole genome duplication in less than 200 million
years (Bowers et al. 2003; Tang et al. 2008; Barker et al. 2009).
In contrast, Vitis appears to have experienced only one duplica-
tion in the past 200 million years (the oldest one in Arabidopsis)
yet has a larger genome with 17 chromosomes (Jaillon et al.
2007). Thus, genome organization and chromosome number is
a product of variation in diploidization processes as well as
genome duplication. Many angiosperm lineages appear to have
experienced two to three rounds of paleopolyploidy in a time
frame of Ks < 2 (e.g., Cui et al. 2006; Barker et al. 2008, 2009).
Considering that only a single duplication event has been
observed in current analyses of fern genomes they may experi-
ence a lower rate of whole genome duplication than
angiosperms.
So, why do homosporous ferns have so many more
chromosomes than angiosperms? One possibility is that
homosporous fern genomes may be less dynamic than angio-
sperm genomes, and may experience chromosomal loss at a
much slower rate. Consistent with this perspective is the
exceptionally low density of genes observed in a fern
15 Karyotype and Genome Evolution in Pteridophytes
Fig. 15.3 Correlation between chromosome numbers and genome size (1C-value in picograms) for angiosperms, gymnosperms, and
pteridophytes. (After Nakazato et al. 2008)
genome (Rabinowicz et al. 2005) and the observation that
genome size and chromosome number are strongly
correlated in ferns (Fig. 15.3; Nakazato et al. 2008). Simi-
larly, earlier observations from cytological studies also
found that fern chromosomes are generally highly uniform
in size and structure across genera (reviewed in Wagner and
Wagner 1980). These observations imply that although fern
genomes appear to lose duplicate genes through gene silenc-
ing, physical genetic material may be lost at a slow rate
compared to many angiosperms and that chromosomal evo-
lution is less dynamic among ferns.
One explanation that may reconcile these observations is
that repetitive elements are less active or abundant in fern
genomes. Repetitive element expansion and contraction has
been long established as a primary determinant of genome
size variation in angiosperms (Leitch et al. 2005), with a
well-established positive correlation of genome size and
numbers of retrotransposons, particularly Long Terminal
Repeat (LTR) retrotransposons (Flavell 1980). Recent
genome analyses in angiosperms, especially grasses, indi-
cate an increase in genome size in the last 10 million years
largely due to transposable element (TE) expansion
(SanMiguel and Bennetzen 1998; Vicient et al. 1999;
Shirasu et al. 2000; Hawkins et al. 2006). These elements
comprise over 60% of large genomes like maize, wheat, and
barley, but less than 50% in rice and about 10% in
Arabidopsis (reviewed in Bennetzen 2002). Although little
is known about the numbers and kinds of TEs in ferns,
reduced copy number of Ty1-copia-like retrotransposons
were recently reported in the homosporous fern Pteris
cretica compared to numbers in other plant species, despite
the fact that it possesses a large genome size and appears to
be a neopolyploid. Thus one possible explanation for the
evolution of stable genome sizes and chromosome numbers
in ferns is suppression of TEs, but this is highly speculative
251
and needs further evaluation. It is not entirely clear why this
would occur, but additional information, including
transcriptome data from more fern lineages, as well as fern
whole genome sequences are needed to determine how, and
ultimately why, fern nuclear genomes are different from
seed plants, and whether such differences are caused by
different genomic compositions or related to homospory
and heterospory.
15.4 Future Directions for Fern Nuclear
Genomics
Furthering our understanding of the evolution of genome
structure and organization of ferns—and vascular plants in
general—will require the application of new technologies as
well as analytical tools. Several new directions have been
initiated in recent years which will be important for the next
generation of advancements. The complete sequence for
a lycophyte, Selaginella moellendorffii, was recently
completed (Banks et al. 2011). Analyses revealed that unlike
angiosperms with low numbers of chromosomes, S.
moellendorffii does not contain evidence of any ancient
polyploidy. This result is consistent with the lower fre-
quency of ancient polyploidy observed in homosporous
ferns relative to angiosperms (Barker and Wolf 2010). Addi-
tional nuclear genomes, as well as phylogenetically diverse
transcriptomes, will undoubtedly be sequenced for ferns and
lycophytes over the next few years and insights into vascular
plant genome evolution will likely come quickly relative to
the past. Combined with novel bioinformatic tools for study-
ing genome evolution, such as chromEvol (Mayrose et al.
2010) and EvoPipes.net (Barker et al. 2010), many of the
twentieth century’s cytological mysteries may finally be
resolved.
252
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 The Incidence of Polyploidy in Natural Plant
Populations: Major Patterns and Evolutionary 16
Processes

Brian C. Husband, Sarah J. Baldwin, and Jan Suda

Contents 16.1 Introduction

16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Polyploidy, the multiplication of complete chromosome sets
16.2 Taxonomic Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
16.2.1 Variation Among Major Plant Groups . . . . . . . . . . . . . . . . . . . 256 per nucleus (n ¼ x in haploid phase, 2n ¼ 2x in diploid
16.2.2 Evolutionary Implications of Taxonomic Variation . . . . . 260 phase, where n is the haploid phase chromosome number
and x is the monoploid chromosome number; Winge 1917;
16.3 Intraspecific Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
16.3.1 Occurrence in Natural Populations . . . . . . . . . . . . . . . . . . . . . . 262 Grant 1981) is a prominent and recurring transition in the
16.3.2 Cytotype Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 evolution of eukaryotic genomes (Otto and Whitton 2000). It
16.3.3 Cytotype Frequencies Within Populations . . . . . . . . . . . . . . . 265 has been documented within every major lineage of
16.4 Mode of Origin (Autopolyploid versus Allopolyploid) 267 eukaryotes (protists, plants, fungi, animals). Among these
16.5 Polyploidy and Life History . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
groups, polyploidy is arguably most prevalent in land plants
16.5.1 Growth Form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 (i.e., angiosperms, gymnosperms, ferns and allies, lycopods,
16.5.2 Reproductive Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 and bryophytes). As in other groups, there is evidence for
16.6 Geographic Distribution, Range Size and Ecological widespread taxonomic heterogeneity in the occurrence of
Adaptation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 polyploidy within plants, and polymorphism at all taxo-
16.7 Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . 271 nomic levels including species and populations (Stebbins
1950; Otto and Whitton 2000). Heterogeneity in the inci-
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
dence of polyploidy has also been associated with geo-
graphic location, environment as well as life history,
morphology and functional traits (Otto and Whitton 2000;
Levin 2002). Collectively, this heterogeneity has fuelled the
long-standing question in evolutionary biology: what
accounts for the evolutionary success of polyploids or,
more specifically, what ecological, phenotypic and genetic
factors promote the formation and establishment of poly-
ploid taxa (Thompson and Lumaret 1992)?
Our understanding of what polyploidy is has broadened
dramatically since its discovery in 1907 (Lutz 1907), a
transformation that has occurred in parallel with advances
in genetic technologies and a better understanding of
genome structure and diversity. Conventionally, polyploidy
is defined as an increase in the number of complete chromo-
some sets relative to the ancestral state. For nearly 100 years,
the primary source of evidence for polyploidy was, by defi-
nition, an increase in chromosome number in multiples of
the base (monoploid) number. The formation of multivalents
B.C. Husband (*)
Department of Integrative Biology, University of Guelph, Guelph,
at meiosis and multisomic segregation of alleles in progeny
Ontario N1G 2W1, Canada are also symptomatic of having additional copies of
e-mail: bhusband@uoguelph.ca

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 255

DOI 10.1007/978-3-7091-1160-4_16, # Springer-Verlag Wien 2013
256
homologous chromosomes and have been important criteria
for identifying certain kinds of polyploids, specifically
autopolyploids (M€untzing 1936; Jackson and Casey 1982).
Autopolyploids are generally defined as those whose
duplicated chromosome sets arise from the same species,
and have a high degree of homology among genome copies,
whereas allopolyploids contain chromosome sets derived
from different species and of weaker or incomplete homol-
ogy (Kihara and Ono 1926; Ramsey and Schemske 1998)
(see Sect. 16.4).
In the era of genomics, the phrase ‘whole genome dupli-
cation (WGD)’ has also been closely associated and used
synonymously with the term polyploidy. This reflects the
addition of a more cryptic form of evidence for polyploidy.
Specifically, phylogenetic, genomic sequence and compara-
tive genomic approaches continue to reveal polyploidy in the
form of multiple copies of whole genome sequences or
genome segments (Soltis and Soltis 1993; see also Fawcett
et al. 2013, this volume). This evidence can occur without a
concomitant duplication in chromosome number, suggesting
that either genomic restructuring and duplication are occur-
ring on a large scale within an existing set of chromosomes
(not polyploidy) or polyploidization has occurred in the
distant evolutionary past and was then followed by a reduc-
tion of chromosomes and/or genes through selection toward
a diploid state (diploidization; Wolfe 2001; Tate et al. 2005).
This reinforces the view that polyploids may fall anywhere
along an evolutionary continuum from initial chromosome
doubling to varying degrees of chromosomal and genomic
restructuring that approaches a more diploid cytogenetic
state. Not all polyploids will follow the same paths. While
many ancient polyploids exhibit multiple gene/genome cop-
ies distributed throughout large portions of their genome,
they will not necessarily exhibit the same consequences as
having multiple chromosome sets (e.g., sterility; Stebbins
1950; Ramsey and Schemske 2002) or double the nuclear
DNA content (Bennett and Leitch 2005), characteristic of
young polyploids. Here, we mostly focus on patterns of
polyploidy as conventionally defined, using chromosome
number as the primary criterion.
After a century of research, our understanding of the
incidence and evolution of polyploids in plants is still in
flux. The early 1900s saw significant advances through the
application of cytogenetic techniques to a wide range of taxa
and consideration of its taxonomic implications. The mid
and late-1900s saw large advances in the spatial scale of
research due to methods such as flow cytometry and growing
interest in the population processes giving rise to polyploids
(Levin 1975; Felber 1991; Thompson and Lumaret 1992).
More recently, comparisons of nucleotide sequences for
large portions of whole genomes have offered a more com-
plete glimpse into the evolution and organization of genome
sequences and their function. Each advance has contributed
B.C. Husband et al.
and altered our inferences about the evolution and evolution-
ary significance of polyploids. Many scholarly reviews on
the incidence of polyploidy have been published in recent
decades with the purpose of exploring specific aspects such
as their taxonomic distribution (Stebbins 1950), modes and
rates of origin (de Wet 1980; Soltis and Soltis 1993; Ramsey
and Schemske 1998; Soltis and Soltis 1999) and impact on
genetic and evolutionary divergence (Otto and Whitton
2000; Soltis and Soltis 2000; Tate et al. 2005).
In this chapter, we examine some of the dominant
patterns of polyploid incidence that have emerged in the
literature but for which detailed explanations are still unre-
solved. These patterns are: (1) variation among taxonomic
groups at or above species, (2) intraspecific variation, (3)
variation in mechanisms of formation (auto vs. allo), (4)
geographic/ecological patterns of polyploid incidence, and
(5) associations between ploidy and life history (reproduc-
tion). Beginning with a summary of the incidence of poly-
ploidy within major groups of plants, we review the state of
evidence with respect to major patterns of incidence and the
progress and challenges associated with understanding their
evolutionary causes.
16.2 Taxonomic Variation
16.2.1 Variation Among Major Plant Groups
One of the most widely recognized patterns in the incidence
of polyploidy in plants is the variation in frequency among
different taxonomic groups (Table 16.1). With increased
scope of sampling and higher throughput analyses, our
estimates have become more complete if nothing else.
From this evidence, it is apparent that polyploidy is widely
distributed but that major plant lineages vary dramatically in
both the frequency of polyploid species and the maximum
ploidy level observed (Table 16.1). In this section, we pro-
vide an up-to-date account of the presence of polyploidy in
major plant groups within the Archaeplastida (sensu Adl
et al. 2005), i.e., the monophyletic clade of primary photo-
synthetic eukaryotes. The Archaeplastida (or plants sensu
lato) comprises three major evolutionary lineages: (1)
Glaucophytes, (2) Rhodophytes (red algae), and (3)
Viridiplantae (green algae plus land plants).
16.2.1.1 Glaucophyta
Glaucophytes are a small group (3 genera, 13 species) of
microscopic algae occurring in freshwater habitats (Keeling
2004). Despite their pivotal role in our understanding of the
evolution of photosynthesis in eukaryotes, little is known
about their chromosome number, ploidy level or genome
size, perhaps at least partly due to the fact that none of the
species is particularly common. Detailed karyological
16 The Incidence of Polyploidy in Natural Plant Populations: Major Patterns and Evolutionary Processes 257

Table 16.1 Frequency of polyploidy, the highest known ploidy level and the record-holder species in different plant groups. The groups do not
represent comparable phylogenetic/taxonomic categories (Data compiled from different sources)
Number of Incidence of Maximum
Plant group extant species polyploidy ploidy level Record-holder
Glaucophytes (Glaucophyta) 13 Unknown Unknown –
Red algae (Rhodophyta) ~6,000 Frequent Moderate Polyides rotundus, n ¼ 6872 (Cole 1990)
(~16-ploid)
Chlorophyta (Green algae s.s.) ~3,800 Frequent Moderate Eraemosphaera viridis, n ¼ 80 (Mainx 1927)
(~20-ploid)
Charophyta (Green algae, ~5,000 (Very) frequent (Very) high Netrium digitus, n ¼ 592 (King 1960)
streptophyte algae)
Liverworts (Marchantiophyta) ~5,000 Rare (~8%) Rather low Riccia macrocarpa, n ¼ 48 (Przywara and Kuta 1995)
(~12-ploid)
Mosses (Bryophyta s.s.) ~12,000 Ambiguous Moderate Leptodictyum riparium, Physcomitrium pyriforme,
(c. 20–80%) (~16-ploid) n ¼ 72 (Przywara and Kuta 1995)
Hornworts ~150 Absent – Anthoceros sampalocensis, n ¼ 10 (Przywara and Kuta
(Anthocerotophyta) 1995)
Lycopods (Lycopodiophyta) ~900 (Very) frequent Very high Huperzia prolifera, 2n ¼ ~556 (Tindale and Roy 2002)
(~50-ploid)
Ferns and allies ~11,000 Very frequent (up Very high Ophioglossum reticulatum, 2n ¼ ~1440 (Khandelwal
(Monilophyta) to ~95%) (~96-ploid) 1990)
Cycads (Cycadophyta) ~250 Absent – Zamia paucijuga, Z. prasina, 2n ¼ 28 (Moretti and
Sabato 1984)
Ginkgo (Ginkgophyta) 1 Absent – 2n ¼ 24
Conifers (Pinophyta) ~550 Very rare (<2%) Low Sequoia sempervirens, 2n ¼ 66 (Hirayoshi and
(hexaploid) Nakamura 1943)
Gnetophytes (Gnetophyta) ~50 Moderate (~30%) Low Ephedra funerea, E. gerardiana, 2n ¼ 56 (Ickert-Bond
(octoploid) 2003)
Monocots ~60,000 Very frequent Very high Voanioala gerardii, 2n ¼ ~596 (Johnson et al. 1989)
(~75%) (~50-ploid)
Dicots s.l. ~200,000 Very frequent Very high Sedum suaveolens, 2n ¼ ~640 (Uhl 1978)
(~75%) (~80-ploid)

investigations of glaucophytes are needed to ascertain
the role of genome duplication in the early evolution of
photosynthetic eukaryotes and the recently sequenced
genome of the glaucophyte of Cyanophora paradoxa
(Price et al. 2012) should shed light on this.
must have occurred independently in several lineages
(Kapraun 2005). In addition, intraspecific ploidy heteroge-
neity recorded in several species (Cole 1990) suggests that
genome duplications have occurred in the recent evolution-
ary history of red algae.

16.2.1.2 Rhodophyta (Red Algae) 16.2.1.3 Viridiplantae (Green Plants)

The red algae (Rhodophyta) are a large and diverse group of
microscopic algae and macroalgae (c. 6,000 species in 700
genera; Kapraun 2005) that mostly occur in marine
environments. Some chromosome counts (Cole 1990) and
estimates of nuclear DNA contents (Bennett and Leitch
2010) are available although still quite fragmentary. Haploid
numbers range from n ¼ 2 to 68–72 (Cole 1990; Kapraun
2005); some orders (e.g., Bangiales) have low chromosome
numbers whereas others have predominantly high numbers
(e.g., Ceramiales, Gigartinales or Gracilariales). Difficulties
establishing the base chromosome number have precluded
precise estimates of the ploidy level, but the maximum value
in the haplophasic gametophytic generation is ~16x (analo-
gous to 32x in groups with predominant sporophytes such as
seed plants). Allopolyploidy has been suggested as a major
evolutionary force in rhodophytes (Nichols 1980) and more
recent investigations show that multiple polyploidy events
Following Lewis and McCourt (2004) and Smith et al.
(2009), the green plants (Viridiplantae) contain two major
clades: (1) Chlorophyta (or green algae sensu stricto),
and (2) Streptophyta. The latter is further divided into
Charophyta (or strephophyte green algae) and Embryophyta
(higher plants), which includes the non-vascular land plants
(bryophytes) and vascular land plants (lycopods, ferns and
allies, gymnosperms, angiosperms), both of which are
karyologically relatively well known.
Green Algae
Green algae are a paraphyletic group of uni- and multicellu-
lar alga with plastids containing chlorophylls a and b. Recent
evidence supports dividing this group into two major clades
(Chlorophyta and Charophyta), which have distinct ecologi-
cal preferences (predominance in marine and freshwater
habitats, respectively; Becker and Marin 2009), plus a
258
residuum of related organisms (Prasinophyta; Lewis and
McCourt 2004; Kapraun 2007). The two major lineages
have differences in genomic properties, particularly in the
extent of polyploidy (e.g., Sarma 1982). A number of tech-
nical challenges to studying their chromosomes (e.g., small
chromosome size, difficulties with sample preparation and in
obtaining suitable material) preclude many general
conclusions.
The Prasinophyta, a paraphyletic and morphologically
diverse group, comprises tiny free-floating microorganisms
with low cellular complexity (Becker and Marin 2009).
Most published genome sizes of prasinophytes are small
enough to suggest that the incidence of polyploidy is
unlikely (range from 0.005 to 0.35 pg/1C, with a modal
value around 0.01 pg/1C; Kapraun 2007). One exception is
Ostreococcus tauri, the smallest free-living eukaryotic
organism. Although the genome is as small as the chloro-
plast genome of other green algae, cytogenetic studies show
that it is fragmented into a comparatively high number of
chromosomes from 14 (Courties et al. 1998) to ~20
(Grimsley et al. 2010), raising the possibility of polyploidy
in prasinophycean green algae.
Although polyploidy has been identified in many groups
in each major lineage, the overall proportion of polyploid
taxa and the maximum ploidy level in the Chlorophyta may
be lower than that of the Charophyta (Kapraun 2005, 2007).
The orders Desmidiales and Zygnematales, which are within
Charophyta, are particularly rich in polyploid species, and
are the most speciose groups of charophytes with approxi-
mately 4,000 described species. The incidence of polyploidy
in these two groups has taxonomic implications in that cell
dimensions are often used as diagnostic criterion for species
delineation. Although difficult to determine, the rate of
interspecific and intraspecific ploidy variation in the
Desmidiales may be quite high (Sarma 1982; Hoshaw and
McCourt 1988), and may be comparable to that of the
angiosperms (see below). One species in this group, Netrium
digitus, is the green alga with the highest known number of
chromosomes. Different strains of this common species were
found to possess from ~30 to ~592 chromosomes in the
haplophase (King 1960; Table 16.1). The Charales consist
of six extant genera with ~400 species (Kapraun 2007),
many of which exhibit polyploidy and have chromosome
numbers up to n ¼ 70 (Sarma 1982). In addition, Charales
possess the largest genome sizes of all algae (up to 19.6 pg/
1C in Chara contraria; Bennett and Leitch 2010), with
values being higher than the upper limit for older lineages
of land plants (e.g., bryophytes). This is particularly impor-
tant for evolutionary interpretations because several
multigene phylogenies have placed Charales as a sister
taxon to land plants (Becker and Marin 2009) although
other molecular studies suggest Zygnematales holds this
position (Wodniok et al. 2011).
B.C. Husband et al.
Bryophytes
Roughly 19,000 bryophytes (i.e., non-vascular land plants)
are classified into three monophyletic lineages, which differ
markedly in the incidence of genome duplication: (1)
liverworts (Marchantiophyta), (2) mosses (Bryophyta), and
(3) hornworts (Anthocerotophyta). Chromosomal data have
been compiled for more than 2,250 species (Fritsch 1991;
Przywara and Kuta 1995), making bryophytes a moderately
well explored plant group from a cytogenetic point of view.
There is a general consensus that genome duplication is
rare in liverworts (~8% of species, plus ~6% that have both
diploid and polyploid cytotypes) and absent in hornworts
(using n > 10 as a threshold for polyploidy; Przywara and
Kuta 1995); the proportion of polyploid mosses is a matter of
debate. In contrast to other bryophytes, mosses show striking
variation in base chromosome numbers, from x ¼ 4 to
x ¼ 19. Assuming that polyploids have gametophytic num-
bers n > 9, Kuta and Przywara (1997) concluded that 84%
of extant mosses have undergone polyploidy and 28% are
considered high polyploids (n > 15). Other authors have
challenged these estimates and suggest that genome dupli-
cation likely plays only a minor role in mosses (i.e., as low as
20% of species). Support for the paucity of polyploids has
come largely from studies of genome size, which reveal a
relatively small overall variation (~12-fold) and relatively
constant values within genera and even families (Voglmayer
2000). Interestingly, the range of genome sizes in liverworts
is much greater, and species with comparatively large
genomes have recently been recorded (Temsch et al. 2010;
see also Leitch and Leitch 2013, this volume). The evolu-
tionary constraints on polyploidy are not clear but may be
linked to strong selection for motile biflagellate male
gametes. The majority of known polyploids in mosses
seem to be of autopolyploid origin although there is growing
evidence that allopolyploids are more frequent than origi-
nally supposed (e.g., Wyatt et al. 1988; Orzechowska et al.
2010) and may even dominate in some groups such as peat
mosses (e.g., Ricca et al. 2008; Karlin et al. 2010). Regard-
less of the frequency of polyploids and mode of origin, the
maximum ploidy in bryophytes is rather low, reaching only
about 16x in haplophase. One of the record-holders is
Physcomitrium pyriforme, a polyploid complex that
encompasses several different cytotypes with up to n ¼ 72
(Table 16.1).
Ferns and Allies (Monilophytes)
The highest incidence of polyploidy among all plants is found
in monilophytes, a monophyletic group comprising five
groups of spore-bearing vascular plants (Psilotales—whisk
ferns; Ophioglossales—ophioglossoid ferns; Equisetales—
horsetails; Marattiales—marattioid ferns; and Leptospor-
angiatae—leptosporangiate ferns), with around 95% of spe-
cies believed to have experienced one or more rounds of
16 The Incidence of Polyploidy in Natural Plant Populations: Major Patterns and Evolutionary Processes
genome duplication in their evolutionary history (assuming
that plants with chromosome numbers above n ¼ 14 are poly-
ploid; Grant 1981). The average gametic chromosome number
in monilophytes (e.g., ~57 in homosporous leptosporangiate
ferns) is higher than in any other group of vascular plants
except lycopods (for example it is just ~16 in angiosperms;
Klekowski and Baker 1966). However, this theory was later
questioned by the results of protein electrophoretic analyses,
which used the number of isozymes per enzyme system as a
proxy for ploidy level (Haufler 1987; Soltis and Soltis 1987).
Despite often very high chromosome numbers, many
leptosporangiate homosporous ferns and horsetails were
found to be genetically diploid. These results led some authors
to conclude that the level of polyploidy in ferns and allies may
in fact be overestimated and supported much more conserva-
tive estimates (e.g., Vida (1976) suggested that just 43.5% of
species were polyploid) (see also Barker 2013, this volume).
The observed incongruence between karyological and
protein electrophoresis data can be explained either by the
origin of monilophytes with high chromosome numbers in
the diploid state or, more probably, by the evolution of this
lineage through several cycles of polyploidy followed by
chromosomal diploidization, gene silencing, and extinction
of diploid progenitors (Haufler 1987). In general, the inci-
dence of polyploidy is higher in groups with subterranean
gametophytes (whisk ferns, ophioglossoid ferns) than those
with photosynthetic and aboveground gametophytes, and
higher in homosporous leptosporangiate ferns than in
heterosporous ones (Klekowski and Baker 1966; Haufler
1987). Some authors have suggested that polyploidy
provides a mechanism for maintaining genetic diversity in
groups with subterranean and spatially isolated monoecious
gametophytes, which have a higher chance of self-
fertilization and increased homozygosity. Perhaps not sur-
prisingly, the maximum ploidy level ever recorded in any
plant is from the pantropical adder’s-tongue species
Ophioglossum reticulatum which may reach up to 96x in
individuals with 1,440 somatic chromosomes (Khandelwal
1990; Table 16.1). Gametic chromosome numbers above
200 (and correspondingly high levels of polyploidy) are
also common in other ophioglossoid ferns and some related
whisk ferns (Brownsey and Lovis 1987). On the contrary,
polyploidy is of lower evolutionary significance (and less
frequent) in heterosporous ferns, which have separate male
and female gametophytes that facilitate out-crossing.
Lycopods
Polyploidy is extensive in the Lycopodiophyta, the
oldest extant lineage of spore-bearing vascular plants
(tracheophytes), which is sister to all other tracheophytes.
Usually, three families are recognized within this lineage, the
homosporous Lycopodiaceae (clubmosses and firmosses) and
the heterosporous Isoetaceae (quillworts) and Selaginellaceae
259
(spikemosses). The distribution of polyploidy in lycopods
generally mirrors the pattern described above for
monilophytes (e.g., higher frequency in homosporous spe-
cies). Considering x ¼ 11 to be the base chromosome number
of the genus Huperzia (L€ove et al. 1977), the level of poly-
ploidy in lycopods may reach ~50x (Table 16.1). However, as
in ferns, several lycopod species have been found to possess
isozyme patterns typical of diploid plants despite their high
number of chromosomes (Soltis and Soltis 1988). Interest-
ingly, aquatic heterosporous quillworts contain more than
60% polyploid species, forming polyploid series ranging
from 3x to 12x, and allopolyploidy is considered to be one of
the major modes of speciation in this group (Troı̀a 2001).
Gymnosperms
The extant gymnosperms (cycads, Ginkgo, conifers, and
gnetophytes) form a diverse but relatively small group of
woody seed-bearing plants with exposed ovules and seeds.
Due to their limited number of species (<1,000), showy
appearance, dominance in many terrestrial ecosystems and
high value in forestry, no other group of plants can compete
with gymnosperms in the wealth of karyological data.
There is compelling evidence that polyploidy is absent in
cycads and Ginkgo and extremely rare in conifers
(Table 16.1). Despite intensive research, only four polyploid
coniferous species, all belonging to family Cupressaceae,
have been found (Khoshoo 1959; Ahuja 2005): three
tetraploids—Fitzroya cupressoides, Juniperus chinensis
‘Pfitzeriana’ and Juniperus squamata ‘Meyeri’, and one
hexaploid—Sequoia sempervirens. While Fitzroya is likely
an autotetraploid, Juniperus chinensis likely originated by
allopolyploidy (the origin of the other two taxa is uncertain).
The lack of polyploidy in gymnosperms may be, at least
partly, explained by their large monoploid genome sizes,
which impose constraints on plant development. Indeed,
experimentally-induced conifer polyploids showed reduced
growth, dwarfism and premature mortality (Ahuja 2005). In
addition, concomitant changes associated with polyploidy
such as increased cell size might not be compatible with
the proper formation of woody fibres, thus constraining the
rise of ploidy level in woody plants in general.
Gnetophytes are unique among gymnosperms in having a
much higher proportion of polyploids (Table 16.1) although
duplicated genomes (based on x ¼ 7) have been observed
with certainty only in the genus Ephedra (Ahuja 2005).
About half of Ephedra species are polyploid but generally
only at the tetraploid level with the exception of octoploid
counts in E. funerea and E. gerardiana (Ickert-Bond 2003).
No obvious geographic pattern exists in the distribution of
diploid and polyploid species, since polyploidy occurs
worldwide. Despite high chromosome numbers in Gnetum
(2n ¼ 44) and Welwitschia (2n ¼ 42) relative to other
gymnosperms, whether these genera are polyploid is
260
unclear. A detailed karyological investigation of the latter
species (Khoshoo and Ahuja 1963) revealed an unusual
chromosome complement consisting of only telocentric
chromosomes, with one pair bearing a satellite, quite dissim-
ilar from that of Ephedra. The high chromosome number in
Welwitschia does not necessarily imply that the species is
hexaploid. In fact, genome sizes of both Gnetum and
Welwitschia are at the lower end of published values for
gymnosperms (Bennett and Leitch 2010; see also Leitch and
Leitch 2013, this volume).
Angiosperms
Chromosome numbers have been determined in about 25%
of angiosperm species (Stace 2000), which provides a solid
basis for spatial comparisons of the incidence of polyploidy
and assessing relationships between genome duplication and
other plant traits (described below). Based on chromosome
number, genome duplication is frequent in both dicots and
monocots (Table 16.1) and can attain high levels, such as
~80x in the dicotyledonous Mexican stonecrop Sedum
suaveolens (Uhl 1978) and ~50x in the monocotyledonous
Madagascan palm Voanioala gerardii (Johnson et al. 1989).
Estimates of the standing frequency of polyploidy vary
from 30% to 80% (Masterson 1994) depending on how they
are calculated. Stebbins (1938) estimated that 20–40% of
angiosperms were polyploid based on the mean proportion
of species per genus that have chromosome numbers that are
more than double the base number of the genus. This esti-
mate is similar to Wood et al. (2009) for vascular plants
(35%), based on current phylogenetic data and similar
criteria. Undoubtedly these values underestimate polyploidy
as they do not consider chromosome multiplication that
occurs in association with the origin of different genera. In
contrast, Grant (1963) assumed that the chromosome base
number for angiosperms was n ¼ 79 and that a species
with n  14 was by definition polyploid. Based on this
premise, he estimated that 57% of species were polyploid.
In turn, Goldblatt (1980) and Masterson (1994) argued that
any organism with n  11 constituted a polyploid, which
amounted to 70% of flowering plants. Otto and Whitton
(2000) estimated the incidence of polyploidy (polyploid
index) based on abundance of haploid chromosomes that
are even-numbered compared to those that are odd-
numbered. From this, they estimated moderate values for
both monocots (31.7%) and dicots (17.7%).
16.2.2 Evolutionary Implications of Taxonomic
Variation
16.2.2.1 Preadaptations for Polyploidy
Heterogeneity in the standing frequency of polyploidy is a
well-established feature of plants, regardless of taxonomic
B.C. Husband et al.
level. Such variation suggests that taxonomic groups have
either different predispositions to polyploid evolution or
different amounts of time to accumulate polyploids (Meyers
and Levin 2006), or both. Heterogeneity among sister clades,
for which divergence time is controlled, suggests that at least
some lineages are preadapted to polyploidization. For exam-
ple, within the genus Chamerion (formerly Epilobium), sis-
ter species C. angustifolium and C. latifolium have both
evolved autotetraploid forms in parallel (Mosquin and
Small 1971), whereas the sister clade, consisting of C.
dodonaei and C. fleischeri, exhibit no polyploidy. Many
such examples exist and raise the question: what attributes
of these species pairs contribute to the difference in poly-
ploidy? The answer may rest with a range of attributes that
influence either the formation of polyploids (e.g., unreduced
gamete production) or their establishment (clonality, self-
fertilization, ecological differentiation). The former are
largely unexamined in natural populations (although see
Bretagnolle and Thompson 1995; Ramsey and Schemske
2002) and the latter are invoked primarily through compara-
tive approaches (Levin 2002), and so the specific traits
involved are rarely unambiguous.
In addition to these specific phenotypic attributes, the
incidence of polyploidy likely varies as a function of intrin-
sic genetic and genomic attributes. For example, Wood et al.
(2009) show clearly that the incidence of polyploidy is
highest in genera with a low base chromosome number
(Fig. 16.1). Similarly, Grif (2000) found a negative associa-
tion between polyploid incidence and genome size. This
pattern suggests that polyploid formation/establishment is
subject to intrinsic constraints determined by the size of
the genome itself. The biological basis for this constraint is
not fully understood but could reflect the increasing negative
effects of high gene dosage, large cell size and increased
meiotic irregularities associated with high genome copy
number. Interestingly, recent genetic analyses of polyploid
yeast indicate that genes controlling mitotic spindle forma-
tion, chromosome cohesion and homologous recombination,
and DNA repair may be particularly important in determin-
ing the stability of polyploids (Štorchová et al. 2006; Thorpe
et al. 2007). Whatever the cause, the negative relation
between genome size and frequency of polyploidy within
genera helps to explain the progressive decline in the inci-
dence of higher ploidy levels on the landscape. This pattern
is clearly depicted in a recent survey of chromosome number
and ploidy in the Californian flora (Fig. 16.2; J. Ramsey and
B. C. Husband, unpublished).
16.2.2.2 Polyploidy and Evolutionary
Diversification
The prevalence of polyploidy within plants has led to debate
about the influence of genome duplication on net species
diversification (Otto and Whitton 2000). Researchers have
16 The Incidence of Polyploidy in Natural Plant Populations: Major Patterns and Evolutionary Processes
Fig. 16.1 Relationship between
the base chromosome number per
genus and the incidence of
Infrageneric Polyploid Incidence (%)
polyploidy (relative to base
chromosome number) in
angiosperms. (From Wood et al.
2009 with permission)
argued, on one hand, that polyploidy may simply accumulate
as a unidirectional mutation with little influence on the rate
or trajectory of evolutionary divergence (Stebbins 1971;
Meyers and Levin 2006). This idea is based on the premise
that ploidy is more likely to increase than decrease in time.
Although reversals do occur, the resulting haploids are rela-
tively rare and often of reduced fitness and high sterility
(Stebbins 1980; Ramsey and Schemske 2002). Alternatively,
polyploidy itself may enhance evolutionary diversification
leading to increased divergence and species richness (Otto
and Whitton 2000). Elevated diversification may occur if
polyploidy accelerates adaptive divergence and evolution of
reproductive isolation (Otto and Whitton 2000) or reduces
species extinction. This divergence may be enhanced
because of the ability to differentiate extra gene copies
(subfunctionalization, neofunctionalization) or the effects
of genomic restructuring on standing genetic and phenotypic
variation (Schranz and Osborn 2004; Tate et al. 2005; see
also Fawcett et al. 2013, this volume).
Several studies have tested for elevated divergence rates
in polyploids by looking for a positive relationship between
the incidence of polyploidy in specific taxonomic groups
(e.g., genera) and species richness. Using this approach,
Petit and Thompson (1999), Otto and Whitton (2000),
Vamosi and Dickinson (2006), and Wood et al. (2009)
found a weak increase in species richness per genus with
the incidence of polyploidy. However, this method
confounds species richness with age of lineage (Meyers and
Levin 2006). Vamosi and Dickinson (2006) controlled for
time by comparing diploid and polyploidy sister taxa in the
Rosaceae and, in that case, found no difference with respect
to species number. Similarly, Wood et al. (2009) compared
the species richness of 59 sister genera, separated by a ploidy
increase, from 34 family-level phylogenies. They, too, found
261
60
a
50
40
b
30
c
20
d
10
0
2n=4-14 2n=16-18 2n=20-24 2n=26-108
Generic Base Count Group
no tendency for polyploid genera to have increased species
diversification. Taken together, the evidence suggests that
although polyploidy may contribute significantly to species
diversification, there is no indication that polyploid lineages
have a higher rate of diversification than non-polyploid
lineages. This result is corroborated by experimental selec-
tion studies, which find that higher ploidy lineages most often
have reduced responses to selection compared to their lower
ploidy progenitors (Zeyl et al. 2003; Anderson et al. 2004).
Quantitative estimates of the contribution of polyploidy to
speciation have been lacking from the literature until recently.
Otto and Whitton (2000) developed a model for estimating
the incidence of polyploidy per speciation event using the
proportion of species with even versus odd chromosome
numbers, expressed as a proportion of the minimum number
of speciation events associated with a chromosome number
change. They estimated that the percentage of speciations
associated with genome duplication is ~7% in ferns and
2–4% in angiosperms. Most recently, Wood et al. (2009)
used 120 phylogenetic trees for angiosperms and 20 trees
for ferns to estimate more directly the rate of polyploid
speciation; they estimated that 15% of angiosperm speciation
events and 31% of ferns are associated with a ploidy increase.
16.2.2.3 Plants versus Animals
Although the incidence of polyploidy in animals is better
understood and recognized as more common than once
thought (Gregory and Mable 2005), it is still clear that the
phenomenon is more widespread in plants, particularly among
sexually reproducing organisms. Why polyploidy is more
prevalent in plants than animals has perplexed biologists and
is still unresolved. The dominant explanations relate to
differences in the likelihood of formation of polyploids or
the potential consequences for the function and survival of
262
Fig. 16.2 Distribution of 1000
somatic ploidy level (2n) among
species of the Californian flora
(N ¼ 3,163 biological species;
346 genera). Ploidy index for
each species is expressed as a 750
multiple of the base chromosome
number for the respective genus.
Frequency
Values range from 2n ¼ 2x to
2n ¼ 20x in the data set but are 500
only shown to 2n ¼ 8x. The
modal peaks illustrate the
prevalence of orthoploid-euploids
compared to anorthoploids and
dysploids or aneuploids among 250
taxa (J. Ramsey and
B. C. Husband unpublished)
0
2n=2.0
polyploid individuals (Gregory and Mable 2005). Jackson
(1976) postulated that triploid infertility posed a barrier to
tetraploid formation in animals. However, triploids are also
prevalent in plants and, in fact, it has been argued they are the
very pathway by which polyploids are produced (de Wet 1980;
Husband 2004). Muller (1925) suggested that polyploidy
interferes with the sex-determining mechanisms, specifically
the sex chromosome : autosome ratios in species where the X
chromosome is dominant. Unfortunately this mode of sex-
determination is not prevalent among animals, and Muller’s
explanation cannot account for the absence of polyploidy in
species with a Y-dominant sex-determining mechanism.
Moreover, Muller’s hypothesis that polyploidy disrupts sex
expression is not corroborated by the abundance of dioe-
cious plant species that contain polyploid series or can be
duplicated experimentally (e.g., Li et al. 2010). Orr (1990)
suggested that in species with a degenerate sex chromosome,
polyploidy disrupts the X : autosomal balance and hence
dosage compensation mechanisms with fatal effects. Plants
are less vulnerable to this because they rarely have degener-
ate sex chromosomes. Thus, while these specific mechanisms
may apply in organisms with dominant X chromosomes and
with degenerate sex chromosomes, it cannot account for the
general scarcity of polyploidy in animals (Gregory and
Mable 2005). Other considerations, such as the effect of
duplication on development, reproductive isolation, the inci-
dence of hybridization and mobility may also be important
(Gregory and Mable 2005).
16.3 Intraspecific Variation
Although polyploidization is often associated with species
diversification (beginning with Hagerup 1927) due to the
barriers to gene flow that result from chromosome
B.C. Husband et al.
2n=3.0 2n=4.0 2n=5.0 2n=6.0 2n=7.0 2n=8.0
Ploidy Index
multiplication, ploidy variation is also observed within tax-
onomic species. The presence of intraspecific ploidy diver-
sity has three main causes. First, genome duplication is an
ongoing genomic mutation within many natural populations
and ploidy polymorphism is therefore expected when poly-
ploid formation is common. In fact, Ramsey and Schemske
(1998) estimated that polyploids form at the rate of 105 per
individual per generation, well above the genic mutation
rate, and in some cases it may be even higher than that
(Husband 2004). Second, depending on the frequency and
pathways of polyploid formation, diploids and polyploids
may be maintained as genetically homogeneous populations,
which are justifiably treated as single taxonomic species.
Finally, ploidy polymorphism may arise as an artefact of
our imperfect taxonomic approach. Since morphological
divergence is often used as a criterion for species delinea-
tion, polyploid lineages may not be recognized at the species
level, especially in autopolyploids, which often have a close
resemblance to their diploid progenitors (Soltis et al. 2007).
In many cases, the patterns of variation within mixed-ploidy
species can provide insights into the early stages of poly-
ploid evolution.
16.3.1 Occurrence in Natural Populations
Intraspecific ploidy variation has been documented in many
plant groups. Based on a broad survey of species, Wood
et al. (2009) reported that 12–13% of angiosperm species
and 17% of fern species are variable for ploidy. Similarly, a
survey of the Californian flora indicated that nearly 13% of
the 2,647 species were polymorphic for ploidy (J. Ramsey
and B. C. Husband, unpublished; Soltis et al. 2007). Intra-
specific ploidy variation has also been observed in most
other plant groups such as red algae (Cole 1990), green
16 The Incidence of Polyploidy in Natural Plant Populations: Major Patterns and Evolutionary Processes
algae (Kapraun 2007), liverworts (Przywara and Kuta 1995),
and mosses (Kuta and Przywara 1997). Surprisingly, ploidy
heterogeneity has even been detected in horsetails (Equise-
tum; Bennert et al. 2005), which have long been a textbook
example of a chromosomally-uniform group. However, to
date, no intraspecific ploidy variation has been detected in
most gymnosperms, with the possible exception of a few
Ephedra species (e.g., E. breana; Khoshoo 1959).
The global frequency of ploidy variation within plant
species is difficult to estimate at this time because the vast
majority of species have not been investigated in enough
detail to confidently describe their ploidy composition. His-
torically, the frequency of intraspecific ploidy variation was
unknown because large-scale karyotypic surveys were time
and cost intensive and not deemed essential for cytotaxo-
nomic treatments. The consequence is illustrated well in a
review of ploidy estimates in 185 species of wild potato
(Solanum section Petota). Hijmans et al. (2007) estimated
that, due to inadequate sampling, only 28% of the mixed-
ploidy species in this group have likely been discovered.
Fortunately, new high-throughput tools, such as flow
cytometry, are enabling more species to be analysed with
much higher sample sizes (Kron et al. 2007). Since the
application of flow cytometry for ploidy determination,
there have been many new observations of additional ploidy
levels. For example, nonaploids have now been detected in
the mostly hexaploid Elytrigia repens and E. intermedia
(9x ¼ 3.8%, 6x ¼ 96.2%, N ¼ 238; Mahelka et al. 2005)
and penta- and heptaploids in the mostly hexaploid Senecio
carniolicus (5x ¼ 1.1%, 7x ¼ 0.3%, 6x ¼ 55.5%,
N ¼ 380; Suda et al. 2007). In the latter study the sample
size has now been increased from 380 to 5,033, and three
additional ploidy levels (3x, 8x, 9x) have been detected,
making S. carniolicus one of the most ploidy-diverse plant
species (2x–9x inclusive; Sonnleitner et al. 2010;
Table 16.2). Overall it is likely that ploidy heterogeneity in
species is underestimated and may continue to increase with
more intensive sampling. Furthermore, because of the varia-
tion in sampling intensity, caution must be taken when
comparing rates of intraspecific polymorphism between
plant groups.
In species with intraspecific ploidy variation, the number
of ploidy states varies from at least two to eight. The distri-
bution of ploidy states across species depends on how the
data were collected. In a survey of chromosome numbers
among polymorphic species of the Californian flora
(N ¼ 334), which is based on moderate sampling per spe-
cies, most species had two ploidy states and the frequency
declined sharply with progressively higher numbers of
ploidy states (Fig. 16.3; J. Ramsey and B. C. Husband,
unpublished). In contrast, using studies of 26 species with
263
more intensive sampling (average sample size of 1,744),
species with three cytotypes were most common and the
frequency decreased less consistently as ploidy number
increased (Fig. 16.3, Table 16.2). The most common two-
cytotype combination was 2x/4x in both surveys while the
most common three-cytotype combination was 2x/4x/6x in
the floristic analysis and 2x/3x/4x in the intensive studies. In
many of the intensive studies, triploids occurred at low
frequencies as in Chamerion angustifolium (3.3%; Sabara
2008), Galax urceolata (11%; Burton and Husband 1999),
Heuchera grossulariifolia (1.4%; Thompson et al. 1997),
and Melampodium leucanthum (0.2%; Stuessy et al. 2004).
One other notable difference between the two datasets was
the high incidence of odd ploidies (3x, 5x, etc.) in the
intensively studied species (84.6%) as opposed to the floris-
tic survey (~4%). This may reflect the increased chance of
detecting rare, odd-numbered ploidies with increased sam-
pling. The prevalence of studies that detect odd-numbered
ploidies in sexual species suggests that intercytotype
hybridization may be more widespread than initially
thought. The low frequencies of such hybrids (Husband
and Schemske 1998; Baack 2004; Trávnı́ček et al. 2010,
2011a; but see Trávnı́ček et al. 2011b) may reflect the
dependence of polyploidy formation on rare unreduced
gametes and the low relative fitness of these ploidies.
16.3.2 Cytotype Distribution
The spatial relationships between ploidy states within spe-
cies can be categorized as sympatric, parapatric or allopatric,
depending on whether they are geographically intermixed,
adjacent or disjunct, respectively. Examples of all three
forms have been described in the literature. One of the
most extreme examples of an allopatric distribution involves
Nymphoides indica. This pantropical species is diploid in the
Old World but tetraploid in the New World (Ornduff 1970).
In contrast diploid and tetraploid cytotypes of the endemic
Galax urceolata have a sympatric distribution throughout
much of the species’ geographic range in the Appalachian
Mountains (Nesom 1983; Burton and Husband 1999). An
example of parapatry is Chamerion angustifolium, which has
diploids in northern latitudes and tetraploids in southern
latitudes in North America and a narrow zone of sympatry
along the southern limit of the boreal forest and in the Rocky
Mountains (Husband and Schemske 1998). Similarly, in
Vicia cracca of Central Europe, diploids in the east give
way to tetraploids in the west (Trávnı́ček et al. 2010).
Geographic patterns of polyploidy may arise through two
main mechanisms. Sympatric and parapatric distributions
can arise when polyploids form de novo from local diploids
264 B.C. Husband et al.

Table 16.2 Summary of 26 intensive surveys of intraspecific ploidy variation. Ploidy variation was surveyed across 2–336 populations and
involved sample sizes between N ¼ 145 and 6,554. Surveys did not always cover the entire geographic range of the species and occasionally
mixed-cytotype populations were sampled more extensively. + indicates that presence of the cytotype was reported but the overall frequency was not
Cytotypes Populations
Species Family N 2x 3x 4x 5x 6x 7x 8x 9x 10x 12x # % odd N % mixed Reference
Actinidia Actinidiaceae 338 64.2 14.8 0.6 20.4 4 0.6 16 E 62.5 Li et al. (2010)
chinensis
Allium Amaryllidaceae 4,347 + + + 3 325 P 23.1 Duchoslav et al. (2010)
oleraceum
Allium Amaryllidaceae 844 26.3 73.7 2 0.0 62 P 6.5 Xie-Kui et al. (2008)
przewalskianum
Arnica Asteraceae 870 43.4 55.3 1.3 3 44.7 21 E 57.1 Kao (2008)
cordifolia
Aster amellus Asteraceae 2,175 34.3 0.1 0.1 65.4 0.1 5 0.3 87 P 6.9 Mandáková and
M€unzbergová (2006)
Arrhenatherum Poaceae 145 72.4 0.7 26.9 3 0.7 2C 50.0 Petit et al. (1997)
elatius
Centaurea jacea Asteraceae 541 41.2 58.6 0.2 3 0.0 26 P 15.4 Hardy et al. (2001)
Chamerion Onagraceae 2,628 53.9 3.3 42.8 3 1.4 51 C 58.8 Sabara (2008)
angustifolium
Dianthus broteri Caryophyllaceae 244 25.8 2.0 41.4 11.1 19.7 5 2.0 25 E 4.0 Balao et al. (2009)
Galax urceolata Diapensiaceae 1,570 55.0 11.0 34.0 3 34.0 42 P 59.5 Burton and Husband
(1999)
Gymnadenia Orchidaceae 3,581 61.7 1.9 35.6 0.5 0.3 5 0.0 43 P 58.1 Trávnı́ček et al. (2011a)
conopsea s.l.
Heuchera Saxifragaceae 855 87.9 1.4 10.7 3 1.4 14 E 21.4 Thompson et al. (1997)
grossulariifolia
Ixeris nakazonei Asteraceae 592 0.7 17.6 3.0 18.3 0.8 59.6 6 4.5 98 E 20.4 Denda and Yokota
(2004)
b b
Knautia arvensis Dipsacaceae 4,648 36.95 0.04 64.01 3 0.04 279 P 6.5 Kolář et al. (2009)
Lythrum Lythraceae 1,884 4.5 0.9 86.2 8.4 4 0.9 124 P 2.4 Kubátová et al. (2008)
salicaria
Melampodium Asteraceae 156 51.3 0.6 48.1 3 0.6 75 E 2.7 Stuessy et al. (2004)
cinereum
Melampodium Asteraceae 440 77.5 0.2 22.3 3 0.2 188 E 2.7 Stuessy et al. (2004)
leucanthum
Oxycoccus Ericaceae 252 17.5 32.1 50.4 3 32.1 11 P 54.5 Suda (2003)
palustris
Parasenecio Asteraceae 271 54.2 0.4 45.4 3 0.4 44 P 4.5 Nakagawa (2006)
auriculata
Pilosella Asteraceae 4,410 26.6 42.6 28.7 1.9 0.2 5 44.5 46 E 23.9 Trávnı́ček et al. (2011b)
echioides
Pilosella Asteraceae 1,059 40.2 36.7 22.8 0.3 4 37.0 336 E 10.6 Mráz et al. (2008)
officinarum
Plantago media Plantaginaceae 569 58.0 0.2 41.8 3 0.2 27 C 33.3 van Dijk et al. (1992)
Ranunculus Ranunculaceae 1,618 42.1 1.2 56.7 3 1.2 38 E 5.3 Baack (2004)
adoneus
Senecio Asteraceae 5,033 33.8 0.1 15.6 0.7 49.6 0.1 0.04 0.1 8 1.0 100 P 45.0 Sonnleitner et al. (2010)
carniolicus
Solidago Asteraceae 500 65.8 11.6 22.6 3 0.0 16 C 50.0 Halverson et al. (2008)
altissima
Vicia cracca Fabaceae 6,554 34.5 0.1 65.4 3 0.1 257 P 7.0 Trávnı́ček et al. (2010)
Mean 1,744 3.7 8.3 90.5 26.6
E
entire range, C contact zone only, P part of range (not specifically the contact zone)
b
Hexa- and pentaploids were also present but they correspond to K. dipsacifolia and K. arvensis  dipsacifolia hybrids, respectively

(primary contact). Alternatively, polyploids may have
diverged elsewhere and, through range expansion, have
come into contact with more distantly related diploids (sec-
ondary contact). These two scenarios cannot be easily dis-
tinguished without additional genetic evidence. In primary
contact zones, plants of different ploidy are genetically more
similar and more closely related to each other than to
geographically distant plants of the same ploidy, whereas
cytotypes in regions of secondary contact are genetically
divergent. A mosaic of both primary and secondary contact
zones has recently been suggested for several species (e.g.,
Rivero-Guerra 2008; Kolář et al. 2009; Trávnı́ček et al.
2010) but only rarely confirmed by molecular techniques
(Kolar et al, 2012). Additional genetic information and
16 The Incidence of Polyploidy in Natural Plant Populations: Major Patterns and Evolutionary Processes 265

0.70
a Galax urceolata
Proportion of species 25

20

15

Percentage of Populations (%)

0.35 10

0
0-20 20-40 40-60 60-80 80-100

b Chamerion angustifolium
0.00 30
2 3 4 5 6 7 8
25
Number of ploidy states
20
Fig. 16.3 Frequency of species containing two to eight ploidy states
in the Californian flora (open bars, N ¼ 334; J. Ramsey and B. C. 15
Husband, unpublished) and among 26 arbitrarily selected species with
intensive sampling (solid bars; studies listed in Table 16.2). Note the 10
large difference in the proportion of species with only two ploidy states 5
observed between the two data sets
0
0-20 20-40 40-60 60-80 80-100

reciprocal transplants would be required to determine what Cytotype Percentage (%)

maintains these distributions and specifically to distinguish
the role of ecological differentiation and environmental
sorting versus selection against hybrids (tension zone).
16.3.3 Cytotype Frequencies Within
Populations
High-throughput ploidy analyses such as flow cytometry are
now enabling intensive population-level surveys, which
have improved detection of rare ploidies, mixed populations
and estimates of cytotype frequencies within populations.
The incidence of mixed-ploidy populations is of particular
interest because it provides insight into the mechanisms of
origin and coexistence of polyploids. Global estimates, how-
ever, are still uncertain because of limited numbers of stud-
ies, heterogeneity in sampling procedures, and complexities
of the geographic structure of species. Based on intensive
geographic surveys of 26 mixed-ploidy angiosperm species
(from 15 families), the proportion of populations with mul-
tiple ploidies per species was variable and ranged from 2.4%
to 62.5%, with the mean of 26.6% (Table 16.2). One exam-
ple is the perennial herbaceous plant, Chamerion
angustifolium. Within the contact zone in the Rocky
Mountains, 59% of populations surveyed contained both
ploidies and several of those had triploid hybrids (Husband
and Sabara 2004; Sabara 2008). In Gymnadenia conopsea,
up to five different cytotypes can coexist within natural
populations (Trávnı́ček et al. 2011a). We note that variation
Fig. 16.4 Distribution of diploid (black bars) and tetraploid (white
bars) relative frequencies among (a) 40 populations of Galax urceolata
in southeastern United States (From Burton and Husband 1999) and (b)
51 populations of Chamerion angustifolium in the Rocky Mountains,
Canada (Sabara 2008). Populations were located within the sympatric
zones of each species
in mixed-ploidy populations differs among species in part
due to variation in the degree of sympatry among cytotypes
and the degree to which sampling was restricted to the
contact zone. Estimates of ploidy mixture are therefore
likely to be overestimates of the species-wide frequency of
mixed-ploidy populations.
How does the incidence of mixed-ploidy populations
compare to theoretical expectations? Theoretical studies
predict that within zones of sympatry, mixed-ploidy
populations (and coexistence generally) should be relatively
uncommon and evolutionary unstable because of frequency-
dependent selection against rare cytotypes (Levin 1975;
Husband 2000). This is because, under random mating,
most potential mates of the rare cytotype are of the alternate
ploidy, yielding unfit hybrids (Felber 1991). This prediction,
however, has rarely been tested in natural populations.
Within the contact zone of Chamerion angustifolium, the
ploidy frequency distribution matches the expected U
shaped pattern. That is, populations are more likely to be
dominated by one cytotype (usually tetraploids, in this case)
than have even frequencies (Fig. 16.4; Husband and Sabara
2004; Sabara 2008). Similarly, in Galax urceolata, evenly
mixed-ploidy populations within the zone of sympatry are
266
relatively uncommon. Most populations are dominated by
one cytotype, occasionally diploids but usually tetraploids
(Fig. 16.4; Burton and Husband 1999).
The presence of mixed-ploidy populations in many
published studies raises the question of what allows
cytotypes to coexist within populations. Theory suggests
that multiple cytotypes may persist if they are prezygotically
isolated by spatial or reproductive segregation (Fowler and
Levin 1984; Rodriguez 1996) or if the rare cytotype forms
recurrently (Felber 1991; Bever and Felber 1992). Evidence
for both processes have been reported. Spatial segregation of
cytotypes due to either ecological differentiation or
environmentally-independent processes such as founder
effects or limited seed dispersal (Kolář et al. 2009) is fre-
quently observed (Bretagnolle and Thompson 1996; Felber-
Girard et al. 1996; Duchoslav et al. 2010; Sonnleitner et al.
2010) and widely considered a key factor in the coexistence
of different ploidies. Polyploidization is often accompanied
by changes in morphology or physiology, resulting in eco-
logical differentiation between diploids and their polyploid
derivatives (Levin 2002). Habitat segregation often occurs at
different spatial scales. On a coarse grain, spatial arrange-
ment of different cytotypes may range from near-complete
niche differentiation (e.g., Felber-Girard et al. 1996) to
apparent sympatry, which may disappear as the scale of
observation becomes finer (Trávnı́ček et al. 2011a, b).
Although spatial segregation is widely observed, more infor-
mation is needed on the influence of this separation on
patterns of prezygotic mating, which is necessary to over-
come minority cytotype exclusion (Husband and Schemske
1995; Husband and Sabara 2004).
In the absence of strong spatial isolation, coexistence may
be favoured by various reproductive barriers that prevent
hybridization between neighbouring cytotypes (Husband
and Sabara 2004). Such barriers can exist at any number of
steps in the reproductive process. Studies have found
differences between ploidy states with respect to insect
visitor composition (Segraves and Thompson 1999) or insect
foraging patterns (Kennedy et al. 2006), which diminish
pollen exchange between cytotypes. This isolation can be
reinforced by flowering asynchrony, associated with
differences in cell cycles and development rates between
ploidies (Bretagnolle and Thompson 1996; Jersáková et al.
2010). Reproductive isolation acting between pollination
and fertilization (gametophytic selection) may also be a
common mechanism to prevent hybridization (Howard
et al. 1998). Reduced germination on stigmata, reduced
pollen tube growth or decreased ovule fertilization of
heterospecific pollen may result in conspecific gamete pre-
cedence (Howard 1999). As demonstrated for Chamerion
angustifolium, conspecific pollen precedence can be strong
and asymmetrical, and may evolve quickly as a direct con-
sequence of polyploidization (Husband et al. 2002; Baldwin
and Husband 2011). Higher rates of self-fertilization in
B.C. Husband et al.
polyploids (Barringer 2007; Husband et al. 2008) compared
with diploids due to the breakdown of self-incompatibility
systems (Stone 2002) and reduced inbreeding depression
(Husband and Schemske 1996) can also reduce extra-ploidy
mating. More studies that quantify the individual and collec-
tive strength of these barriers are needed to understand their
role in promoting coexistence of multiple ploidies.
Coexistence will also be promoted by the recurrent for-
mation of polyploids within diploid populations (Felber
1991), although in this case polyploids are usually
maintained at low frequencies. Triploids have been reported
at low levels in several diploid species (e.g., Bureš et al.
2004; Koutecký 2007; Slovák et al. 2009; Dušková et al.
2010). Although there is evidence that polyploidy has
repeatedly evolved in some taxa (Soltis and Soltis 1999), it
is unlikely that the frequency is high enough to fully com-
pensate for the mating disadvantage experienced by rare
cytotypes (Ramsey and Schemske 1998). Until now, there
is only one case (Pannonian populations of Hieracium
echioides) in which complex ploidy mixtures have clearly
been maintained by a sufficient frequency of polyploid
origins (Trávnı́ček et al. 2011b). Diploid-polyploid coexis-
tence may be further enhanced in species with dominant
mechanisms of clonal reproduction (Eckert et al. 2003;
Joly and Bruneau 2004; Kao 2007), allowing them to bypass
or further diminish the mating disadvantage experienced by
sexual species (Levin 1975).
Coexistence is particularly common in species in which
diploid-polyploid hybrids are produced (Table 16.2). In
these cases, coexistence may occur because the hybrids are
partially fertile and serve as a regular source of higher ploidy
states through backcrosses to diploid progenitors (Bever and
Felber 1992; Husband 2004). Coexistence is evolutionarily
stable particularly when the hybrid polyploid (most often 3x)
produces monoploid and polyploid (unreduced) gametes,
which can result in the production of both diploid and
polyploid progeny. Few direct tests of this possibility exist
for natural populations (although see Husband 2004). Alter-
natively, mixed populations in these species may simply be
transient stages in the evolutionary transition from diploid to
polyploid. In the absence of triploids, the evolutionary tran-
sition from diploid to tetraploid is very rapid once the
conditions for the spread of tetraploids are met, indicating
that mixed-ploidy transition states will be rare on the land-
scape (Fig. 16.5). However, the same transition with fertile
triploids present is more complex (Fig. 16.5). Mixed
populations will be more common and will take several
forms. Based on simulations (Fig. 16.6), populations often
enter a brief stage of 2x/3x followed by longer periods in 2x/
3x/4x, 3x/2x/4x, 4x/3x/2x, and 4x/3x stages before becoming
fixed for 4x. Two species in which triploids are present show
high frequencies of mixed-ploidy populations and patterns
of cytotype structure consistent with this transition. Galax
urceolata contains a modest number of 2x/3x (15%) and 3x/
16 The Incidence of Polyploidy in Natural Plant Populations: Major Patterns and Evolutionary Processes 267

Fig. 16.5 Transition states in the a

evolution of tetraploid 2x 2x, 4x 4x
populations from diploids in (a)
(ancestral)
absence of triploids, (b) presence
of triploids. Bold arrows indicate
where the presence of polyploids 2x, 3x, 4x
can accelerate their own b
establishment in a positive 2x 2x, 3x 2x, 3x, 4x 3x, 4x 4x
feedback. Dotted arrows indicate
(ancestral)
that these stages may occur as 2x, 4x 2x, 4x
stable states under some
conditions

4x (5%) populations, but the majority of mixed populations viewed as less common and less important evolutionarily.
are 2x/3x/4x (27.5%) (Burton and Husband 1999). In This perception has prevailed from the early 1900s
Chamerion angustifolium, 1.9% of populations were 2x/3x, (M€untzing 1936), as a result of the success in synthesizing
no populations contained 3x/4x and 12.5% were 2x/3x/4x new allopolyploids or recreating de novo existing polyploid
(Sabara 2008). Unfortunately few species have been exam- species. Further, researchers such as Wettstein (1927) and
ined within this evolutionary framework. others argued that autopolyploids were not viable and would
eventually revert to diploids. In contrast, Blakeslee and
Avery (1919), and Jorgensen (1928) have argued that
autopolyploids can be viable under certain circumstances
16.4 Mode of Origin (Autopolyploid versus
but are likely to be rare in nature and necessarily associated
Allopolyploid)
with apomixis and vegetative reproduction. Stebbins (1950)
and Grant (1981) have also argued that autopolyploids
Polyploids are generally classified as being one of two types:
should be rare as a result of abnormal chromosome pairing
autopolyploids and allopolyploids (Kihara and Ono 1926).
and chromosomal segregation leading to infertility of
Autopolyploids are formed through the multiplication of
gametes and inviability of offspring. Ramsey and Schemske
genomes within species, either through the union of genomes
(1998) estimated key parameters in the formation of
from different individuals or a single individual.
polyploids and found that the rate of formation is likely to
Allopolyploids represent those formed through the union of
be higher in hybrids, thus providing an additional argument
chromosome sets from different species either by
for the prevalence of allopolyploids.
hybridization between autopolyploids or chromosome dou-
The relative frequencies of auto- and allopolyploids
bling of diploid hybrids. Distinguishing these forms has never
remain unresolved, not only because of the difficulty in
been straightforward since by this definition, allopolyploids
classifying polyploids but also because of difficulties
can vary widely with respect to the homology of parental
detecting them. Techniques for characterizing patterns of
chromosome sets and patterns of inheritance (Ramsey and
genetic inheritance were not readily available until the adop-
Schemske 1998). With respect to the genetic similarity among
tion of isozyme analyses in evolutionary biology. Moreover,
genomes, polyploids vary along a continuum from homoge-
the close morphological similarities between autopolyploids
neous (autopolyploidy), to partially divergent (segmental allo-
and their diploid progenitors have made some taxonomists
polyploidy) and highly divergent genomes (allopolyploidy)
reluctant to recognize them as distinct species (Soltis et al.
(Stebbins 1947). Typically, autopolyploids are associated
2007). Thus, most autopolyploids are hidden as cryptic spe-
with multivalent formation and polysomic inheritance,
cies within diploid taxa. Nevertheless, over time, as detec-
whereas allopolyploids exhibit bivalents and disomic inheri-
tion improves, the general consensus is that autopolyploids
tance (Stebbins 1947, 1950; Jackson and Casey 1982; Ramsey
may be found to be more common than once thought (Soltis
and Schemske 1998), although exceptions are not uncommon.
and Soltis 1993, 1999; Tate et al. 2005).
The inconsistencies in the genetic attributes of auto- and
allopolyploids create difficulties in classifying polyploids
unambiguously. This is further exacerbated by the fact that
diploid progenitors are often not known or may be extinct. 16.5 Polyploidy and Life History
Phylogenetic reconstructions of taxonomic groups are offer-
ing a method for delineating them that is in parallel to the 16.5.1 Growth Form
definition currently used (Rousseau-Gueutin et al. 2009).
Plant biologists have long disagreed over the relative Current evidence suggests that the frequency of polyploidy
frequency and evolutionary importance of auto- and differs among different life forms. The proportion of
allopolyploids. Historically, autopolyploids have been polyploids is generally highest among perennial herbaceous
268
Fig. 16.6 Change in frequency
of diploids, triploids and
tetraploids during a simulated
transition from a diploid
population to tetraploid. In this
individual-based simulation,
diploids and tetraploids had equal
Cytotype proportion
fitness but triploid fitness was at
10%. Diploids produced 0.03
unreduced gametes per
generation. Mixed populations
persist through most of the 130
generations required for
tetraploids to become fixed
plants, compared to annuals, bulbs and woody species
(Stebbins 1938). This was confirmed in several early vege-
tation surveys in Canada (Clark et al. 1909), Switzerland
(Gustafsson 1948), and Pakistan (Baquar 1976), which
revealed that perennials were more likely to be polyploid
and annuals more likely to be diploid. In addition, Otto and
Whitton (2000) report that the high rate of polyploidy in
dicots is driven by polyploidization in herbaceous species
and not woody ones. However, little experimental research
beyond comparative surveys has explored the basis for these
patterns. It has been suggested that reduced frequency of
polyploidy in trees may be the result of the impact of cell
size on the formation of wood fibres, as with gymnosperms,
while polyploidy in bulbs may be constrained by large
monoploid genome sizes (Otto and Whitton 2000). The
low frequency of polyploidy in annuals may be because of
strong selection against polyploid establishment due to the
frequency-dependence of homoploid mating and the impor-
tance of rapid development associated with colonizing spe-
cies (Leitch and Bennett 2007). Until analyses control for
phylogenetic trends we cannot be sure that this pattern is
consistent across groups. In fact, Vamosi and Dickinson
(2006) recently found no association between ploidy and
herbaceous habit in the Rosaceae family.
16.5.2 Reproductive Systems
Since the early twentieth century, plant biologists have
debated the association between polyploidy and reproduc-
tive systems (e.g., Ernst 1918; Gustafsson 1947; Stebbins
1950). Since then, particular views have been perpetuated
but without a full systematic analysis. Here we examine the
B.C. Husband et al.
1
0.8
Diploid
Triploid
Tetraploid
0.6
0.4
0.2
0
0 20 40 60 80 100 120 140 160 180 200
Generations
relationship between polyploidy and two related aspects of
reproduction: asexual reproduction (including clonality and
apomixis) and self-fertilization.
16.5.2.1 Asexual Reproduction
Wettstein (1927) hypothesized that polyploidy was only
possible in plants with apomixis or vegetative reproduction.
Gustafsson (1948) was the first to survey different floras for
the incidence of asexual reproduction in polyploid plants and
he found very strong trends. Surveys of polyploidy in
animals have also provided evidence for the association
between polyploidy and asexual reproduction. In fact, a
leading hypothesis for the deficiency of polyploidy in
animals is the scarcity of asexual reproduction (Schultz
1969) and in support of this is the observation that poly-
ploidy is present among animal groups capable of asexual
reproduction such as molluscs (Burch 1964), flatworms
(White 1954), and insects (Lokki and Saura 1980). Thus,
the association between asexuality and polyploidy is consis-
tent between plant and animal kingdoms.
Apomixis and Polyploidy
Many plants that reproduce by apomixis (embryos that form
asexually) are polyploid (Stebbins 1941). In fact there are
many examples of mixed-ploidy species, such as Pilosella
officinarum (Mráz et al. 2008), that are comprised of apo-
mictic polyploids and sexual diploids. Polyploidy and apo-
mixis may co-occur because they share common
developmental pathways. Both traits may be induced after
hybridization, involve the production of unreduced gametes,
and restore fertility after hybridization. Although progress
has been made in identifying the genetic determinants of
apomixis in many cases (Nogler 1984), understanding when
16 The Incidence of Polyploidy in Natural Plant Populations: Major Patterns and Evolutionary Processes
these traits are selected and how they facilitate polyploid
formation and establishment has not been fully explored.
A reasonable explanation for the link between apomixis
and polyploidy is that the production of unreduced gametes
is required for both phenomena. In gametophytic apomixis,
the most common form of apomixis in polyploids (Stebbins
1950), unreduced egg cells develop into functional embryos
(parthenogenesis) that are genetically identical to the mater-
nal plant. Similarly, the most likely mode of polyploid
formation is via fertilization involving at least one
unreduced gamete (Ramsey and Schemske 1998).
Unreduced gametes may also facilitate the transmission of
the genes coding for apomixis. This could occur if the genes
are lethal in the haploid form (Nogler 1984) or if the genes
are associated with deleterious recessive mutations as in
Hieracium (Bicknell et al. 2000) and as inferred in
Taraxacum (van Dijk 2003).
The concurrent evolution of apomixis and polyploidy may
also be associated with their connection to hybridization. Both
traits can be induced after hybridization and may be selected
to restore fertility after hybridization. Ernst (1918) first pro-
posed that hybridization could trigger the production of both
polyploidy and apomixis. This could occur since diploid
hybrids suffer from numerous meiotic disturbances, and
such dysfunction can lead to the production of unreduced
gametes. The frequency of unreduced gametes has been
shown to be higher in hybrids than non-hybrid diploids
(Ramsey and Schemske 2002) and this leads to the testable
prediction that apomixis is more common in allopolyploids
than autopolyploids (Whitton et al. 2008), or in offspring from
inter-ploidy crosses than from single cytotype crosses. Apo-
mixis and polyploidy may also both be favoured to restore
fertility after hybridization or in other cases when sexual
reproduction is less effective (e.g., mate limitation and/or
when there is a high risk of inbreeding depression). Even if
apomixis arises equally in diploid and polyploid populations,
it may only become fixed when sexual reproduction is mal-
adaptive (Stebbins 1950; Dickinson et al. 2007).
Vegetative Reproduction (Clonality)
As mentioned previously (Sect. 16.5.1), polyploids are more
likely to be perennial and diploids more likely to be annual.
This difference becomes more pronounced in clonal, or
“root-wandering” perennials, which are about twice as likely
to be polyploid than annual (reviewed by Gustafsson 1948).
There are three main explanations for the correlation
between polyploidy and clonality, the relative importance
of which has not been fully assessed.
First, genome duplication may directly trigger the devel-
opment of modes of vegetative reproduction that weren’t
present in the diploid progenitor (M€ untzing 1936) or may
intensify the extent of clonality (Stebbins 1950). This effect
might be expected if genome duplication results in an
269
increased number of meristems per plant followed by differ-
entiation into meristems with a diverse range of functions.
Second, asexual reproduction may be prevalent in
polyploids because diploid species with clonal reproduction
are more apt to facilitate the formation and establishment of
polyploid populations (Stebbins 1938; Bretagnolle et al.
1998; Otto and Whitton 2000). This is because clonality
enables the polyploid to persist in the face of the strong
mating disadvantage experienced by newly formed
polyploids (i.e., minority cytotype exclusion: Levin 1975;
Otto and Whitton 2000) and increases the availability of
polyploid mates.
Lastly, vegetative reproduction may be intensified rela-
tive to diploids through selection after the genome duplica-
tion event (Stebbins 1950). A disproportionate increase in
clonality of polyploids might be favoured by selection in
mixed-ploidy populations to reduce the effects of
hybridization and minority cytotype exclusion. Currently,
however, there are few empirical tests of differences in the
degree of asexual reproduction between diploid and poly-
ploid relatives. One such example is Campanula patula in
which the proportion of vegetative reproduction via runners
(and plant longevity) increases from diploids to octoploids
(F. Rooks, H. Látalová, J. Suda and F. Krahulec, unpub-
lished). There is a need for comparisons of vegetative repro-
duction among diploids, initial polyploids and established
polyploids within and among sister taxa to isolate the effects
of genome duplication, preadaptation and selection on the
evolution of vegetative reproduction in polyploids.
16.5.2.2 Polyploidy and Mating System
The association between polyploidy and mating system in
angiosperms is supported by two apparently conflicting
patterns. On one hand, there is a significant tendency for
polyploids to be more self-fertilizing than diploids (Barringer
2007; Husband et al. 2008). On the other, polyploidy has been
shown to be associated with gender dimorphism and hence
outcrossing in several genera (Miller and Venable 2000). The
linkage to self-fertilization was originally identified by Grant
(1956) and Stebbins (1957) who observed a higher incidence
of allopolyploids among lists of selfing species. More
recently, Barringer (2007) conducted the first phylogen-
etically informed analysis of variation in outcrossing rate (t)
estimates using genetic markers to determine the proportion
of progeny derived from cross-fertilization. Based on 235
taxa, he identified 32 phylogenetically independent contrasts
in which increased ploidy was associated with a decrease in
outcrossing rate (and hence increase in self-fertilization).
Husband et al. (2008) used 10 diploid-polyploid congeneric
species pairs to address the same question. Like Barringer,
they found that polyploids were significantly more selfing (or
less outcrossing; t ¼ 0.23) than diploids (t ¼ 0.51). They also
discovered that increased selfing (lower outcrossing) was
270
primarily associated with allopolyploids (t ¼ 0.20), not
autopolyploids (t ¼ 0.64). This pattern corroborates
Stebbins’ (1957) original observations, although broader sam-
pling is needed to confirm the difference between types of
polyploids across angiosperms.
The correlation between ploidy and selfing has been
predicted on multiple grounds. Grant (1956) and Stebbins
(1957) suggested, and Ramsey and Schemske (1998)
demonstrated, that polyploid formation is more likely via
selfing than outcrossing, especially in allopolyploids (34
times more likely). Once formed, the establishment of poly-
ploid populations is also aided by selfing since the strong
negative fitness costs of between-ploidy mating are avoided
(Levin 1975). In addition, most theory argues that the fitness
costs of selfing (i.e., inbreeding depression) should be lower
in polyploids (Lande and Schemske 1985; Hedrick 1987;
Ronfort 1999; Rausch and Morgan 2005; but see Busbice
and Wilsie 1966), making it easier for selfing to evolve.
These predictions are supported to different degrees by a
limited number of empirical studies (Husband and Schemske
1997; Rosquist 2001; Barringer and Geber 2008).
In contrast to this body of evidence, Miller and Venable
(2000) showed that, in multiple plant families, there was a
significant association between increased polyploidy and the
shift from hermaphroditism to gender dimorphism. This
pattern suggests that, in fact, outcrossing can be favoured
in some polyploids (or polyploidy is favoured in outcrossed
populations). Miller and Venable (2000) have proposed that
polyploidy creates the conditions for the spread of male
sterile mutants in hermaphrodite populations by (1) causing
the loss of self-incompatibility, (2) increasing the rate of
self-fertilization, and (3) expressing sufficient inbreeding
depression in hermaphrodites to favour the establishment
of females. This hypothesis requires inbreeding depression
to be strong in new polyploids, a prediction that is not
supported by observations of synthesized polyploids (Hus-
band et al. 2008). However, it is possible that inbreeding
depression rises in tetraploids with successive generations of
selfing, as recently observed by Ozimec and Husband
(2011). Clearly, additional work on a variety of species is
required to reconcile the strongly opposed patterns of mating
in diploids and polyploids and to understand the evolution-
ary forces driving outcrossing.
16.6 Geographic Distribution, Range Size
and Ecological Adaptation
A major observation made by plant biologists over the last
century concerns the association between polyploidy and the
geographic and ecological range limits of species (Levin
2002). A species’ geographic range, the area within which
an organism occurs for part of its life, is the result of many
B.C. Husband et al.
complex interacting forces, including the history of coloni-
zation and the contemporary interactions between the
organisms and its environments. Polyploidy is considered
an important genetic determinant of a species’ range. Indeed,
arguably, this single genetic event may represent the largest
influence on the ecological or geographic range of a species.
In general, plant biologists have postulated that: (1) poly-
ploid ranges will differ from those of the diploids, (2) poly-
ploid species will have larger geographic ranges, and (3) the
ranges for polyploid species will include more extreme
environments such as high latitudes and altitudes, than dip-
loid species (Hagerup 1927; Stebbins 1950, 1985; Otto and
Whitton 2000; Levin 2002). The differences in geographic
range between polyploids and diploids have been explained
through a variety of different mechanisms (M€untzing 1936;
Stebbins 1950). For example, polyploid individuals are
expected to have greater breadth of ecological tolerance, or
plasticity, due to higher heterozygozity and allelic diversity
(genetic or biochemical buffering) or greater adaptive poten-
tial due to higher population genetic diversity (Otto and
Whitton 2000). They may also be less vulnerable, at least
initially, to the maladaptive effects of gene flow from other
diploid populations (Kirkpatrick and Barton 1997). Finally,
polyploids may differ phenotypically from diploids in a
systematic way, as a result of the direct effects of genome
duplication, particularly cell size. On this basis, it has been
argued that polyploids will be more cold tolerant, drought
tolerant or tolerant of disturbance (Hagerup 1927). The
effects of these different mechanisms are rarely disentangled
but they might be expected to influence species ranges and
ecological amplitude in different ways.
The strength of the link between distribution and ploidy is
as varied as the kinds of evidence used to assess it. In the first
half of the twentieth century, the dominant evidence was in
the form of floristic and taxonomic case studies (L€ove and
L€ove 1943; Clausen et al. 1945) that examined whether
the frequency of polyploids was higher in extreme
environments, be it high latitude (Tischler 1935; L€ ove and
L€ove 1943; Packer 1969), altitude (Sokolovskaya and
Strelkova 1940), or saline habitats (Shimotomai 1933;
Tischler 1937). In general, these studies led to the conclu-
sion that polyploidy is more common in extreme
environments, which was often attributed to greater cold-
hardiness and tolerance to long day photoperiods than
diploids. In recent years, high-throughput analyses of poly-
ploidy have been extended into the arctic (Brochmann et al.
2004) and less-studied regions at lower latitudes (Popp et al.
2008; Suda et al. 2009; Dušková et al. 2010); similar latitu-
dinal comparisons have been conducted in mosses (Kuta and
Przywara 1997). In general, the high incidence of polyploidy
at high latitude holds reasonably well (Suda et al. 2009).
The use of floristic approaches for testing the effect of
polyploidy on distribution has been criticized for a number
16 The Incidence of Polyploidy in Natural Plant Populations: Major Patterns and Evolutionary Processes
of reasons. First, the comparison of floras is confounded with
different biogeographical and phylogenetic histories
(Gustafsson 1947, 1948). The prevalence of polyploids in
northern floras may be directly related to the species that
colonize those areas and their characteristics (e.g.,
perenniality, evergreenness) rather than selection favouring
polyploids per se. Secondly, there is much inconsistency in
the geographic position of diploids and polyploids within
individual taxonomic groups as well as in their cold hardi-
ness, when tested experimentally (Bowden 1940; Gustafsson
1947; Nielsen 1947). Finally, geographic regions have not
received uniform effort when screening for polyploids, par-
ticularly subtropical and tropical regions. What has been
lacking until recently is a broad comparison of range
attributes of a large number of species, ideally where the
range attributes of polyploid species were compared to sister
diploid species or at least those of recent common ancestry.
Three studies have compared the range attributes of
diploids and polyploids in broad surveys. Petit and
Thompson (1999) compared the ecological range limits of
diploids and polyploids in the Pyrenees. They found no
evidence that polyploids occurred over a wider range of
ecological environments than diploids. Similarly, Stebbins
and Dawe (1987) found little effect of polyploidy on the
percentage of species that were widespread in European
genera. In a recent analysis, Martin and Husband (2009)
compared the extent and mean geographic and ecological
ranges for diploid and polyploid taxa within 144 North
American plant genera. They found no difference in the
geographic area of the range between ploidy levels and no
evidence that the centroid of the range of polyploids was
shifted further north than diploids. Interestingly, they did
observe that range overlap between congeneric diploids
and polyploids was greater than between two congeneric
diploids. This requires further examination but could reflect
the ability to coexist because of strong ecological or repro-
ductive isolation. In addition, comparisons of the breadth,
maximum or minimum range values for precipitation or
temperature showed no consistent differences between
ploidy levels (Martin and Husband 2009).
Potentially contrary to this evidence is a parallel literature
that has used comparative evidence to show that the inci-
dence of polyploidy increases with invasiveness and
decreases with rarity (Pandit 2006; Pandit et al. 2011). The
most recent study, based on an analysis of 640 endangered
species and 81 invasive species, found that rare species are
most likely to be diploid, whereas invasive species are more
likely to be polyploid. To the extent that invasive species
tend to be widespread, this pattern may conflict with the lack
of association between range size and ploidy. However,
there may be no conflict as definitions of invasive/rare take
many factors into account, only one of which is geographic
271
range size, and may also be influenced by variation in politi-
cal designations and lack of uniformity of information on
which designations are based.
The literature to date does not imply that polyploidization
has no effect on ecological tolerances or species ranges.
Indeed, there is a rich literature corroborating the effect of
genome duplication on habit, phenotype and physiology
(Levin 2002). Polyploids frequently differ in geographic
range from diploid relatives. However, the data show, in
general, that differences among polyploids and their
congeners are not systematic in direction and may not be
greater than the differences among homoploid congeners.
Nevertheless, the question remains as to what drives the shifts
in range size between individual diploids and polyploid
relatives. Are these shifts a direct result of genome duplication
or are they the product of evolutionary divergence operating
after duplication? Work on the ecological attributes of newly
synthesized polyploids will be particularly insightful in this
regard. For example, research on the distribution of diploid
and tetraploid Chamerion angustifolium (fireweed) supports
the idea that range shifts are not achieved as a direct result of
the duplication (Sabara 2008). In fireweed, diploids are more
often found at higher altitudes and latitudes than tetraploids.
Reciprocal transplants show that populations are weakly
differentiated and adapted with respect to elevation. However,
in terms of traits such as drought physiology and reproductive
morphology, neopolyploids resemble diploids more than
extant tetraploids (Sabara 2008; Maherali et al. 2009),
suggesting that genome duplication is not the sole contributor
to the patterns of ecological divergence.
16.7 Conclusions and Future Directions
Our knowledge of the incidence of polyploidy is dominated
by a limited number of long-standing patterns first identified
by the pioneers in this field. These patterns describe varia-
tion in polyploidy as a function of taxonomic group, life
history traits and geography. In recent years, additional
evidence has accrued using broader taxonomic perspectives
and new genetic or phylogenetic analyses, but how have our
perceptions of polyploidy and its significance changed?
Although our perception of the dominance of polyploidy in
plants has remained relatively constant, some notable changes
to the patterns have occurred. With higher throughput
analyses, we have seen a steady increase in the frequency of
polyploidy, with almost no group untouched, and an increase
in the incidence of autopolyploidy and intraspecific ploidy
variation. Combined with recent genomic comparisons,
which confirm a role of genome duplication in early plant
evolution, we can only conclude that polyploidy has
influenced almost all extant species at some stage in their
272
evolutionary history. This observation has led to more signifi-
cant questions such as: does polyploidy contribute to evolu-
tion in a way that diploids do not (Otto and Whitton 2000)?
The incidence of polyploidy, combined with a phylogenetic
perspective, has been equivocal on its role in species diversi-
fication, but for the first time we have estimates of the rate of
polyploid speciation (Wood et al. 2009). With respect to
geography, the dogma that polyploids have larger ranges and
are more common at high latitudes and extreme environments
has become less clear and possibly even misleading since
phylogenetic control and broad comparative analyses have
been used. Similarly, patterns of reproductive biology among
polyploids are variable and even conflict with common dogma.
The original goal of studying the incidence of polyploidy
was to identify key traits or attributes that may offer the
universal key to polyploid significance. However, as more
evidence and more powerful analyses accumulate, the
strength of particular associations is not always clearer. In
fact, if there is one lesson emerging, it may be that poly-
ploidy does not have a single cause or unifying advantage.
Rather the impact of polyploidy on ecological and evolu-
tionary function is consistent only in that it has an effect,
even if the specific direction is unpredictable. This suggests
that it may be time to redirect our focus towards the context
in which polyploidy operates. This approach shifts the
emphasis from asking whether polyploids will differ from
diploids to what will best predict the effects of genome
duplication in any one circumstance. An understanding of
phenotypes at the level of gene interactions and function will
be essential for building this predictive framework.
To continue to advance our understanding of the patterns of
occurrence of polyploidy in plants, several avenues of research
would be profitable. In some less intensively studied taxo-
nomic groups (e.g., Glaucophyta), there is still a need for
additional surveys of ploidy. Flow cytometry and whole
genome sequencing should help to achieve this. In addition,
it is clear that certain traits such as those related to the forma-
tion of polyploids (e.g., unreduced gametes) have not been
explored in natural populations fully enough to draw any
inferences (Bretagnolle and Thompson 1995). In many cases,
however, the data are now widely available in the literature
and in accessible databases (Bennett and Leitch 2010). The
challenge now will be to re-evaluate our long-accepted
patterns within a phylogenetic framework. This will provide
a stronger measure of the phylogenetic contributions to poly-
ploid formation and establishment but will also allow us to test
patterns with more rigour and generality (e.g., Vamosi and
Dickinson 2006; Wood et al. 2009). Lastly, experimental
research will be critical for corroborating our hypotheses
with more direct approaches. For example, the use of synthetic
polyploids (neopolyploids), in conjunction with physiological,
genetic and genomic tools, will be instrumental for evaluating
what of these many associations are the direct effects of
B.C. Husband et al.
genome duplication as opposed to selection operating after-
wards (Bretagnolle and Lumaret 1995; Ramsey and Schemske
2002), and more generally what are the evolutionary processes
underlying the patterns of polyploid incidence in plants.
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 Significance and Biological Consequences
of Polyploidization in Land Plant Evolution 17
Jeffrey A. Fawcett, Yves Van de Peer, and Steven Maere

Contents 17.1 Introduction

17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
17.2 Detecting and Dating Ancient WGDs Using Genomic
The abundance of polyploids in nature has been long
Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 recognized. However, it is only in the last decade with the
17.2.1 Detecting Ancient WGDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 advance in genome sequencing that we have begun to appre-
17.2.2 Dating Ancient WGDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 ciate their evolutionary potential. In fact, some researchers
17.3 Molecular Biological Consequences of Polyploidy . . . 281 considered polyploidy as an ‘evolutionary dead end’ that
17.3.1 Genetic and Epigenetic Changes . . . . . . . . . . . . . . . . . . . . . . . . . 281 cannot result in any further diversification of species
17.3.2 Changes in Expression Pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
(Stebbins 1950). It therefore came as a big surprise that the
17.3.3 Adaptive Potential of Novel Polyploids . . . . . . . . . . . . . . . . . 283
17.3.4 Evolution of Polyploid Genomes; Divergence and genome of Arabidopsis thaliana, the first plant genome to be
Homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 sequenced, chosen for its small genome size, had experi-
17.4 Evolutionary Significance of WGDs in Angiosperms 285 enced multiple rounds of polyploidization in its evolutionary
17.4.1 Genome Duplications and Speciation . . . . . . . . . . . . . . . . . . . . 285 past (Arabidopsis Genome Initiative 2000). This finding
17.4.2 Reciprocal Gene Loss and Subfunction Partitioning . . . . 285 brought about some new questions; when did these Whole
17.4.3 Dosage Balance Effects and the Regulatory Spandrel . . 286 Genome Duplications (WGDs) occur? Are there traces of
17.4.4 WGDs and Evolutionary Novelty . . . . . . . . . . . . . . . . . . . . . . . . 286
17.4.5 WGDs Increase Evolutionary Potential, and May Do So ancient WGDs in other plant genomes? And most impor-
for a Long Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 tantly, what has been the significance of these polyploidy
17.4.6 Opportunities, Niches and Mass Extinctions . . . . . . . . . . . . 288 events in plant evolution?
17.5 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . 288 Subsequent analyses of various other plant species over the
past decade, based on whole-genome sequences or large
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Expressed Sequence Tag (EST) collections, have to a certain
extent answered these questions. We now know that most, if
not all eudicots are derived from a hexaploid ancestor
(Fig. 17.1) (Jaillon et al. 2007; Tang et al. 2008a, b). The
ancestor of Arabidopsis has undergone two WGDs after the
hexaploidization (Jaillon et al. 2007; Ming et al. 2008; Tang
et al. 2008a). Other lineages such as those of poplar, tomato,
and apple have also each undergone additional WGDs (Tuskan
et al. 2006; Tang et al. 2008a; Velasco et al. 2010). The lineage
of grass species such as rice, maize, sorghum, and wheat appear
to have undergone two WGDs after the divergence of
monocots and eudicots, although the timing of the earlier
WGD is less clear (Tang et al. 2010). Furthermore, it was
S. Maere (*)
recently suggested that one WGD has occurred in the common
Department of Plant Systems Biology, VIB, Technologiepark 927, ancestor of all seed plants, and that another WGD has occurred
Ghent B-9052, Belgium in the common ancestor of all angiosperms (Jiao et al. 2011).
Department of Plant Biotechnology and Bioinformatics, Ghent Also, the genome of the moss species Physcomitrella patens
University, Technologiepark 927, Ghent B-9052, Belgium has experienced a WGD (Rensing et al. 2007; 2013 this
e-mail: stmae@psb.vib-ugent.be

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 277

DOI 10.1007/978-3-7091-1160-4_17, # Springer-Verlag Wien 2013
278 J.A. Fawcett et al.

monocots

Liriod
eudicots

Persea
endro
magnoliids

s
Acoru
S ar

sa
um

n
Nu

Mu
Am

a
a

yz
ph
bo m

Or
ar
Gy re ghu
mn lla or
os S
pe
rm a
s Ze
olzia
hsch
Esc
Glycine
Actinidia

Medicago Camellia
s
Malu Sola
num
lix Ge
Sa rbe
He ra
s
lu lia
pu nt
Po hu
a

La
sic

ctu
Viti
is
as

Gossy
e
ops

Carica

ca
Br

s
Cleom
bid

pium
Ara

Fig. 17.1 Phylogenetic tree of angiosperm species showing ancient
WGDs that have been proposed (Lysák et al. 2005; Cui et al. 2006;
Jaillon et al. 2007; Barker et al. 2008, 2009; Lescot et al. 2008; Fawcett
et al. 2009; Paterson et al. 2009; Schnable et al. 2009; Schmutz et al.
2010; Shi et al. 2010; Tang et al. 2010; Velasco et al. 2010). Ancient
WGDs are observed throughout angiosperms, including in the common
ancestor of most (if not all) eudicots. Blue dots represent duplication
events whereas blue stars represent triplication events. Although many
genera contain species with different ploidy levels (e.g., Solanum,
Oryza, Gossypium), these polyploids are not shown. The red
line represents the Cretaceous - Tertiary (Paleogene) boundary.
Although there is some uncertainty associated with the ages of the
WGDs, it seems that many of the WGDs occurred close to the time
of the K-T extinction event (Fawcett et al. 2009)

volume). Note that the main focus of this chapter will be on

angiosperms as little is known about WGDs in plant species
outside angiosperms, with the exception of Physcomitrella.
‘How many percent of plants are (ancient) polyploids?’
has been a question that many researchers have been trying
to answer (Otto and Whitton 2000). This question is becom-
ing increasingly irrelevant as in fact most angiosperms, or
even most seed plants might have a polyploid ancestry
(Soltis et al. 2009; Jiao et al. 2011). The significance of
polyploidy in the evolution of angiosperms is now undeni-
able, and a good understanding of polyploidy is vital if we
are to understand the evolution of angiosperms. In this
chapter, we will first describe how ancient WGDs can be
identified in the genomes of extant species, and how the
timing of ancient WGDs can be determined. After that, we
will describe the molecular biological consequences of
polyploidization: what happens to the genes and genomes
of polyploid species immediately after polyploidization, and
how do the genes and genomes evolve? Finally, we will
discuss how the different WGDs might have contributed to
the evolutionary success of the descendant plant lineages.
17.2 Detecting and Dating Ancient WGDs
Using Genomic Data
17.2.1 Detecting Ancient WGDs
Ancient WGDs can be detected within the genomes of extant
species by two different approaches—based on the age dis-
tribution of duplicated genes, and based on genomic collin-
earity. If the species in question had undergone a WGD in its
evolutionary past, the genome would contain a large number
of duplicated gene pairs of the same age. Thus, if one were to
build an age distribution of a large collection of duplicated
gene pairs, a large-scale or whole-genome duplication
should correspond to a significant peak in the age distribu-
tion (Fig. 17.2). The number of synonymous substitutions
per synonymous site (KS) is often used as a proxy for time
since duplication, assuming that synonymous substitutions
occur at a constant rate across all duplicates. The KS of
duplicate pairs created by the same WGD should be similar.
The KS distribution approach has the advantage of not
17 Significance and Biological Consequences of Polyploidization in Land Plant Evolution 279

Full paranome Transcription regulators

1000 80
900 70
α
800
60
Arabidopsis thaliana

700
600 β, γ hexaploidy 50
500 40
400 30
300
20
200
100 10
0 0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

4000 300
3500 p
250
Populus trichocarpa

3000
200
2500
2000 150
1500 γ hexaploidy
100
1000
50
500
0 0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

2500 120

100
2000
ρ
80
Oryza sativa

1500
60
1000 σ?
40

500
20

0 0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Ks

Fig. 17.2 In KS distributions of duplicated gene pairs, genome
duplications (arrows) show up as peaks superimposed on a roughly
exponentially decaying background of small-scale duplications. Due to
increasing variation in KS with age since duplication, older peaks
become progressively broader and may appear merged with other
peaks. On the left are the KS distributions of the whole paranome
(complete set of duplicated genes) in three plant species. On the right
are the corresponding distributions for transcription regulatory genes
only. Even without rigorous modelling, it is clear from these figures
that WGDs contributed comparatively more to the expansion of tran-
scription regulatory gene families than to the expansion of the whole
paranome. Distributions were calculated as in Maere et al. (2005).
WGD nomenclature follows Tang et al. (2008a, 2010). The older s
WGD proposed to have happened in monocots (Tang et al. 2010) is not
clearly visible, and neither are the recently proposed angiosperm and
seed plant WGDs (Jiao et al. 2011)

requiring assembled genomic sequences, and many putative WGDs in many lineages such as rice, tomato, soy-
researchers have used it to search for traces of ancient bean, lettuce, and California poppy (Blanc and Wolfe 2004a;
WGDs in EST libraries, leading to the identification of Schlueter et al. 2004; Cui et al. 2006; Barker et al. 2008).
280
One downside of the KS approach is that if the WGD is too
old, the KS values of the duplicate pairs have a large vari-
ance, and the WGD peak may overlap with other peaks or
even an artefactual saturation peak in the KS distribution
(Fig. 17.2). In such cases, more sophisticated approaches
such as building age distributions based on phylogenetic
dating are required (Fawcett et al. 2009; Jiao et al. 2011).
WGDs that are too young can also escape detection as the
divergence between the duplicates will be low and indistin-
guishable from the young single gene duplicates or from
sequencing errors, which occur frequently in ESTs (Blanc
and Wolfe 2004a). It is worth noting that segmental duplica-
tion or bursts of single gene duplications can also be respon-
sible for peaks in the age distribution (Blanc and Wolfe
2004a). It is advisable to be cautious when the number of
duplicates present in the peak is small and/or if the KS of the
peak is large and perhaps saturated, even if it is statistically
significant by some measure.
If the whole genome sequence is available, ancient
WGDs can be detected based on genomic collinearity (Van
de Peer 2004). Although the similarity between the
duplicated genomes decreases as they diverge from each
other and many duplicate genes are lost, remnants of genome
duplications are often still detectable in the form of
duplicated blocks, i.e., sequence stretches containing
members of the same paralogous gene pairs in the same
(collinear) order, intermingled with single-copy genes that
have lost their WGD paralog. Different methods exist to
assess the statistical significance of a collinear arrangement
of duplicated gene pairs. Including additional genomes in
collinearity analyses can increase the resolution of detecting
duplicated blocks. Genes that have lost their WGD paralog
in one species might still have a paralog in another species,
allowing for transitive detection of collinear regions
(Vandepoele et al. 2002; Proost et al. 2009). If several
paralogous regions appear to be of similar age, i.e., contain
paralogous gene pairs with similar KS, we can assume that
they were created by the same WGD event.
Collinearity analyses on various recently sequenced plant
genomes have indeed uncovered several instances of ancient
WGDs. The genome of rice showed evidence of an ancient
WGD that has been confirmed to have occurred in the
common ancestor of most grass species such as rice,
maize, sorghum, and wheat, ~65–70 Mya, in addition to an
older WGD (Paterson et al. 2004; Tang et al. 2010). The
genomes of the legumes Medicago, Lotus, and soybean
showed that a WGD occurred before the divergence of
these three species (Cannon et al. 2006; Sato et al. 2008;
Schmutz et al. 2010). The genomes of poplar and apple both
contain evidence of independent WGDs that occurred >60
Mya and >50 Mya, respectively (Tuskan et al. 2006;
Velasco et al. 2010).
J.A. Fawcett et al.
17.2.2 Dating Ancient WGDs
Once ancient WGDs are identified in a number of species,
the next task is to identify the timing of these WGDs. For
instance, is an ancient WGD identified in one species shared
with another species or specific to that species? Again,
genomic collinearity analysis has proven to be very power-
ful. The detection of collinearity between multiple eudicot
genomes, including genomes that appear to have a more
ancestral genome structure such as grapevine and papaya,
substantially improved our understanding of the timing of
ancient WGDs in eudicots (Tang et al. 2008a; Van de Peer
et al. 2009b). Grapevine belongs to an early-diverging line-
age of rosids and is thus sister to both Arabidopsis and poplar
(Jansen et al. 2006). It was observed that many regions of the
grapevine genome showed homology to two other regions
elsewhere in the genome, representing a triplicate structure.
This suggests that the grapevine lineage derived from a
hexaploid ancestor. In addition, some of these regions
showed a one to two relationship to regions in the poplar
genome, and a one to four relationship to regions in the
Arabidopsis genome. This led to the suggestion that the
hexaploidization occurred in the common ancestor of grape-
vine, Arabidopsis, and poplar, and that one additional WGD
occurred in the lineage leading to poplar, and that two
additional WGDs occurred in the lineage of Arabidopsis
(Jaillon et al. 2007). The genomes of papaya and apple
also showed traces of this hexaploidization event,
confirming a common hexaploid ancestry of most rosids
(Ming et al. 2008; Velasco et al. 2010). The genome of
papaya, which is a rosid species more closely related to
Arabidopsis than to poplar or grapevine, had no traces of
additional WGDs after the hexaploidy event, indicating that
the two additional WGDs observed in the Arabidopsis
genome occurred after the divergence of the Arabidopsis
and papaya lineages (Ming et al. 2008). Comparison of the
genomic sequences of grapevine and tomato, an asterid
species, suggests that tomato also shares the hexaploidy
event (Tang et al. 2008b). This would mean that the
hexaploidization pre-dates the divergence of rosids and
asterids, and that it would thus be shared by most eudicots.
In the absence of multiple genomic sequences, phyloge-
netic approaches have been used to identify the timing of
WGDs. Suppose that we want to ask whether a WGD that
has been detected in the genome of species A is shared with
species B or not. If the WGD had occurred in the ancestor of
A after the divergence of A and B, the paralogs derived from
the WGD (A1 and A2) should be more closely related to each
other than to their closest homolog in B (say B1), leading to
the topology ((A1,A2),B1). On the other hand, if the WGD
had occurred in the common ancestor of A and B, the closest
homolog in B should be the ortholog of A1 or A2 and more
17 Significance and Biological Consequences of Polyploidization in Land Plant Evolution
closely related to A1 or A2 than A1 and A2 are to each other,
leading to the topology ((A1,B1),A2) or ((A2,B1),A1).
Although this is a useful approach, it can easily yield
misleading results because the different substitution rates in
different lineages can result in erroneous topologies, and
careful choice of species and tree reconstruction is required
(Tang et al. 2008b; Van de Peer et al. 2009b; Jiao et al. 2011).
Ideally, one would like to know the timing of any WGD
event in terms of ‘how many million years ago’, rather than
‘before the divergence of these species and after the diver-
gence of these species’, because this will enable us to place the
WGDs in the context of the geological time frame. The sim-
plest approach to dating WGDs is to build an age distribution
based on the KS of all WGD-derived duplicate pairs, and
convert the KS value of the peak of the distribution into time
based on estimates of synonymous substitution rates.
Although such an approach can provide an adequate approxi-
mate in some cases, it is also known to be error-prone for
various reasons. For instance, the substitution rate is known to
vary across time and species, and estimating the rate for any
given species can be very difficult—consider how our under-
standing of the evolutionary rate of the model species
Arabidopsis thaliana has totally changed in the past few
years (Koch et al. 2000; Jakobsson et al. 2006; Beilstein
et al. 2010). In addition, dating older events is especially
problematic because the KS value becomes less reliable and
may approach saturation. Recent studies have attempted to
date WGD events by building phylogenetic trees with gene
families including WGD-derived duplicates and dating the
divergence of all duplicated genes using methods that account
for rate variation such as Bayesian methods or the penalized
likelihood method (Fawcett et al. 2009; Jiao et al. 2011). Such
phylogenomic-like approaches will increase in accuracy as
genomic data from many different species become available.
17.3 Molecular Biological Consequences
of Polyploidy
Although several groups of species contain recently formed
polyploids and evidence of ancient WGDs has been found in
all angiosperm genomes, the process leading from the for-
mation of polyploids to their establishment is rather com-
plex. First, we will discuss the changes that occur in
polyploid genomes immediately or shortly after polyploi-
dization. Recent natural polyploids in genera such as Bras-
sica, Arabidopsis, cotton, soybean, wheat, tobacco, and
Tragopogon can provide insights into the early stages of
polyploid evolution (Wendel 2000; Kashkush et al. 2003;
Comai 2005; Doyle et al. 2008; Jackson and Chen 2010). In
addition, synthetic polyploids have been created in many of
these species, which allow us to trace the changes that occur
immediately after the polyploidization.
281
17.3.1 Genetic and Epigenetic Changes
Various genetic changes such as indels and rearrangements
have been reported to occur shortly after polyploidization in
both synthetic and natural polyploids. In synthesized
allopolyploids in wheat, elimination of DNA sequences
was observed already in the first generation (Ozkan et al.
2001). Although genetic changes were found to be rare in the
first generation of ~50 re-synthesized allopolyploid lines of
Brassica napus, several genetic changes, in particular non-
reciprocal transpositions of DNA segments between
homoeologous chromosomes were documented during the
S2–S5 generations (Gaeta et al. 2007; Lukens et al. 2006).
Tragopogon mirus is an allopolyploid that has formed
repeatedly in nature within the last 80 years (Soltis and Soltis
1999). Loss of DNA has been observed in multiple
individuals of this species that are of independent origin
(Koh et al. 2010). On the other hand, no large-scale genomic
rearrangements were observed in the synthetic Gossypium
allopolyploids, suggesting that the responses vary among
different polyploids (Liu et al. 2001). The genetic changes
appear to be a combination of random and non-random
changes. In the ~50 re-synthesized allopolyploid lines of
B. napus, there was a large variation among the different
lines of the extent and direction (in which parental genome
the changes took place) of the genetic changes. However, the
changes were not random in respect to the genomic location,
and tended to occur more frequently on chromosomes with
large regions of homeology between the two subgenomes
(Gaeta et al. 2007).
Changes at the epigenetic level, such as DNA methyla-
tion or histone methylation and acetylation, are also likely to
play a significant role in newly formed polyploids (Lukens
et al. 2006; Wang et al. 2006b). Changes in the pattern of
DNA methylation have been especially well-studied. Syn-
thetic polyploids in wheat, B. napus, and Arabidopsis
suecica all showed non-additive methylation patterns with
respect to their diploid progenitors, but to varying degrees.
No methylation changes were observed in synthetic cotton
allopolyploids. Methylation changes have also been
documented in natural polyploids in wheat and Spartina
anglica (Shaked et al. 2001; Salmon et al. 2005). Extensive
changes in DNA methylation were also documented in the
first generation of the aforementioned synthetic B. napus
allopolyploids, in contrast to the paucity of genetic changes
that took place in the first generation (Lukens et al. 2006).
Many of these methylation changes remained fixed in their
S5 progeny, although some methylations were reversed and
some new changes were observed (Gaeta et al. 2007).
Another documented phenomenon following polyploidy
is the activation of transposable elements (TEs). This is most
probably related to changes at the epigenetic level
that reverse the epigenetic silencing of TEs. Increased
282
transcriptional activity of TEs following polyploidy is
thought to be common and has been documented in various
polyploids including synthetic allopolyploids in wheat and
Arabidopsis (Kashkush et al. 2003; Madlung et al. 2005;
Parisod et al. 2010). TE amplification after polyploidization
appears to be less common, although it has been documented
in some cases such as the Tnt1 retrotransposon in Nicotiana
synthetic allopolyploids (Petit et al. 2010). This is likely to
be because while TE activation is more of an immediate
response to the effect of hybridization or polyploidization,
the amplification of TEs requires their fixation in new geno-
mic loci, which might be countered by purifying selection
due to deleterious effects. Nevertheless, the doubling of the
genome might relax the purifying selection against deleteri-
ous TE insertions, and reduction in the effective population
size following polyploid formation might increase the
chance for neutral or slightly deleterious TE insertions to
fix through stochastic processes, thereby providing the
opportunity for TE amplification (Parisod et al. 2010).
17.3.2 Changes in Expression Pattern
One question of great interest has been how the expression
patterns change in homoeologous genes following polyploi-
dization (Hegarty and Hiscock 2008; Jackson and Chen
2010). For instance, are the expression levels of both
homoeologous genes maintained at the same levels as in
their parental diploid species? How do the expression
patterns of homoeologous genes diverge?
It has been shown in various polyploids that the expression
levels of homoeologous genes are frequently non-additive,
meaning that one or both genes are up- or down-regulated in
the polyploid species compared to their parental species.
In Arabidopsis synthetic polyploids formed by combining
A. arenosa and A. thaliana, 5–38% of the homoeologous
gene pairs were non-additively expressed. More than 65% of
these genes were down-regulated compared to the parental
midpoint, and >94% of the repressed genes were genes
that were expressed at higher levels in A. thaliana than in
A. arenosa, indicating extensive suppression of A. thaliana-
derived homoeologs in the allopolyploid (Wang et al. 2006b).
Similar biases where homoeologs from one of the parental
species get preferentially silenced have also been observed in
cotton and Brassica (Chen and Pikaard 1997; Flagel et al.
2008). A recent study quantified the leaf transcriptome size of
a Glycine tetraploid species and its diploid progenitors.
Despite the genome size of the tetraploid being close to
the sum (94.3%) of the genome sizes of the two diploid
progenitors, the transcriptome size was only 70% of the
sum of the transcriptomes of its diploid progenitors,
suggesting global downsizing of the transcriptome (Coate
and Doyle 2010).
J.A. Fawcett et al.
The expression pattern of homoeologous genes can also
diverge in a tissue-specific manner. For instance, in some
homoeologs in cotton natural or synthetic allopolyploids, the
A-genome homoeolog would be silenced in some tissues
whereas the D-genome homoeolog would be silenced in
other tissues (Adams et al. 2003; Chaudhary et al. 2009).
Novel expression patterns have also been observed where a
homoeolog is expressed in a tissue in which it was not
expressed in the diploid progenitor (Chaudhary et al.
2009). Such expression profile changes are also often
observed in F1 diploid hybrids, and it has been suggested
that the merging of two different genomes, rather than the
doubling, has the largest effect on the expression divergence
(Chaudhary et al. 2009). This is not to say that genome
doubling does not affect the expression pattern, as shown
in Senecio (Hegarty et al. 2006). Interestingly, the expres-
sion changes of some homoeologs in synthetic allopoly-
ploids mimic the expression changes observed in natural
allopolyploids that have in some cases undergone a few
million years of evolution (Chaudhary et al. 2009). This
suggests that tissue-specific differential silencing that occurs
immediately after polyploidization might be maintained for
over a million years. A recent study showed that the expres-
sion of small RNAs also changes in allopolyploids. It was
suggested that the small RNAs might have a role in adjusting
the expression levels of mRNAs and maintaining genome
and chromatin stability (Ha et al. 2009).
The various genetic and epigenetic changes described
above can partly explain the expression divergence that
occurs in polyploids. For instance, indels or mutations
including TE insertions, or changes in DNA methylation in
the upstream regulatory region are likely to alter the expres-
sion pattern of the downstream genes. It has indeed been
shown that blocking DNA methylation can reactivate
silenced genes in polyploids (Lee and Chen 2001). However,
expression divergence has been observed when little genetic
or epigenetic changes have been detected, and it is thought
that the coming together of genes with diverged regulatory
sequences plays a large role (Albertin et al. 2006; Chaudhary
et al. 2009; Hegarty et al. 2006). For instance, consider two
genes A and B in two different species 1 and 2; in the diploid
parents, A1 regulates the expression of B1 and A2 regulates
the expression of B2, and the expression pattern of B1 and
B2 may be different due to divergence between the parent
species (at the genetic or epigenetic level). When the two
different species form a new polyploid (or a hybrid), the
polyploid can potentially gain two new regulatory
interactions where A1 regulates B2 and A2 regulates B1,
so that B1 and B2 can be regulated by both A1 and A2. Thus,
B1 and B2 can have novel expression patterns that were not
present in either parental species (Fig. 17.3). Such reuniting
of diverged regulatory interactions is likely to be responsible
in part for the immediate expression changes and novel
17 Significance and Biological Consequences of Polyploidization in Land Plant Evolution
A1 B1
A1
A2
Fig. 17.3 The emergence of novel expression patterns in newly
formed polyploids. Gene A regulates the expression of gene B in two
different species 1 and 2, but the genes A and B have independently
accumulated changes in both species since their divergence. As such,
the expression pattern of gene B (B1 and B2) is different in the two
species. This can be due to changes at the genetic or epigenetic level in
the coding or non-coding regions of A and/or B. As these two species
expression patterns that are induced by genome merger
(Riddle and Birchler 2003).
17.3.3 Adaptive Potential of Novel Polyploids
Polyploidy can be generally considered an abnormal state
that has many disruptive effects. Most of the molecular
biological consequences described above are usually delete-
rious (Comai 2005). However, some of these changes might
also result in new traits that allow polyploids to adapt to
environments that their diploid progenitors could not
(Hegarty and Hiscock 2008). In fact, it has been frequently
observed that polyploids have colonized novel, often harsh
environments (Fawcett and Van de Peer 2010). In Achillea
borealis, autohexaploids have a fitness advantage over
tetraploids in dune habitats. Transplantation experiments
showed that neohexaploids already exhibited higher survi-
vorship than tetraploids in dune habitats, indicating that an
increase in ploidy level per se can result in a fitness advan-
tage (Ramsey 2011). The adaptive potential of polyploids
has been questioned because deleterious mutations will per-
sist longer in a polyploid population and increase the genetic
load, whereas the effects of new beneficial mutations might
get masked by other alleles at the same locus (Stebbins
1971). However, theory suggests that polyploid populations
283
A2 B2
B1
B2
form an allopolyploid, apart from inheriting the regulatory interactions
from both parental species (A1–B1 and A2–B2), the polyploid can
potentially gain novel regulatory interactions (A1–B2 and A2–B1)
immediately. This can result in phenotypes not present in the parental
species, which might allow the polyploids to adapt to different
environments
may adapt faster than diploid populations under certain
conditions. For instance, the masking of deleterious
mutations can provide polyploids an initial advantage over
diploids as long as the deleterious allele is recessive and the
masking is stronger in the polyploid than in the diploid (Otto
and Whitton 2000). This might contribute to the survival of
polyploids shortly after polyploidization by buffering crucial
functions (Chapman et al. 2006). Also, new advantageous
alleles can spread more rapidly in polyploid populations if
the allele is dominant and if the population size is small
(Otto and Whitton 2000).
The ability of polyploids to adapt to more extreme
environments is probably related more to gene expression
changes after polyploidization than merely to copy number
changes (Crow and Wagner 2006; Ha et al. 2007; Hegarty
and Hiscock 2008; Hegarty et al. 2008; Jackson and Chen
2010). Novel regulatory interactions that emerge due to the
hybridization of two genomes can cause immediate changes
in gene expression and concomitant changes in phenotype.
One example is flowering time variation across Arabidopsis
diploids and tetraploids. In Arabidopsis, the flowering
time is largely controlled by the FRIGIDA (FRI) and
FLOWERING LOCUS C (FLC) genes where FRI
upregulates FLC expression that inhibits early flowering
(Johanson et al. 2000). FRI is non-functional in the early-
flowering ecotypes of A. thaliana, whereas the late-flowering
284
autotetraploid A. arenosa contains a functional FRI. By
contrast, the FLC gene is intact in A. thaliana, whereas
A. arenosa has two copies of FLC whose promoter regions
contain deletions (Wang et al. 2006a). The natural allotetra-
ploid A. suecica, a winter-annual whose parental species
are A. thaliana and autotetraploid A. arenosa, exhibits an
extremely late flowering time, even later than A. arenosa. It
was found that synthetic F1 allotetraploids containing the
genomes of A. thaliana and A. arenosa already showed a
later flowering time than A. arenosa autotetraploids. This
was attributed to the trans-activation of the stronger
A. thaliana-derived FLC by the functional A. arenosa-derived
FRI, which allowed higher expression of FLC than their
parental plants. Thus, it appears that the novel regulatory
interaction that arose in the allopolyploid due to the diver-
gence between the two parental species resulted in a more
extreme phenotype, providing the allopolyploid an immedi-
ate adaptive advantage in novel environments (Wang et al.
2006a). More generally, the merger of divergent genomes
through polyploidization can lead to transgressive segrega-
tion, i.e., the formation of phenotypes that are more extreme
than the diploid parent phenotypes, and hybrid vigor
(Osborn et al. 2003; Rieseberg et al. 2003, 2007; Crow and
Wagner 2006; Hegarty et al. 2008).
Polyploids may also support a wider range of transgressive
and non-transgressive expression patterns than their diploid
counterparts, through an increase in the number of possible
allele dosage combinations (Osborn et al. 2003). This may
facilitate adaptation to novel environments (Hegarty and
Hiscock 2008) if there is sufficient allelic variation in the
polyploid population. Allelic variability may be accomplished
through gene flow between polyploid populations that
originated independently (Soltis and Soltis 1999), through
mutations and epigenetic changes that occur immediately
after polyploidization, or through polysomic inheritance in
autopolyploids (Soltis and Soltis 2000; Osborn et al. 2003)
Epigenetic changes are also likely to be important in the
adaptation of polyploids. Epigenetic changes can result in
gene expression changes, and are likely to proceed more
rapidly than genetic changes, creating phenotypic variation
in the early stage of the evolution of polyploids. A recent
study on three closely related allopolyploid species of the
orchid Dactylorhiza suggested that the ecological differenti-
ation and adaptation of the different species were facilitated
by methylation changes (Paun et al. 2010). The authors
showed that the three species, and also two geographically
distinct populations of D. traunsteineri, could be clearly
differentiated based on their methylation pattern (Paun
et al. 2010), even though the genetic divergence between
the different allopolyploid species is limited (Pillon et al.
2007). Moreover, the gene expression differences between
the allopolyploid species were best explained by differences
in their methylation pattern.
J.A. Fawcett et al.
17.3.4 Evolution of Polyploid Genomes;
Divergence and Homogenization
As the polyploid species continues to evolve, the two
duplicated genomes usually diverge further from each
other by accumulating various changes at the genetic level.
Although epigenetic changes and expression changes might
be crucial in determining adaptation and short-term success
of polyploids, the genetic changes are likely to have a greater
impact on an evolutionary time scale (Edger and Pires 2009;
Kejnovsky et al. 2009; Fawcett and Van de Peer 2010). DNA
loss, rearrangements, recombination between homoeologous
regions, and TE amplification result in the disruption of
synteny between the two genomes. A large number of redun-
dant genes is lost or pseudogenized, causing the genome to
return to a more diploid state. But the genes that are retained
in duplicate play a crucial role in the evolutionary success of
polyploids, as we discuss below.
Although it is often assumed that the divergence between
paralogous sequences increases over time, this is not always
true. Non-reciprocal recombination, or gene conversion can
occur between paralogous sequences, which will counteract
their divergence. Homogenization by gene conversion has
been reported in young polyploids such as cotton and tobacco
(Kovarik et al. 2008; Salmon et al. 2010). In addition, exten-
sive gene conversion has been recently reported in duplicates
created by the WGD that occurred in the common ancestor of
most grass species such as rice and sorghum. In the most
extreme case, the short arms’ termini of rice chromosomes
11 and 12 show very high similarity throughout the entire
3 Mb, including non-coding regions, and was initially thought
to represent a <10 million year old segmental duplication (The
Rice Chromosomes 11 and 12 Sequencing Consortia 2005;
Wang et al. 2005). However, this duplication was found to be
present also in sorghum, which diverged from rice ~50 Mya,
and other grass species (Jacquemin et al. 2009; Paterson et al.
2009; Wang et al. 2009). Thus, it appears that this duplicated
segment was created by the WGD that occurred in the com-
mon ancestor of rice and sorghum, and has been extensively
homogenized in rice since then. The corresponding segments
in sorghum also appear to have undergone gene conversion,
but to a lesser extent than in rice. Gene conversion, like any
other mutational process, can have both positive and negative
effects (Fawcett and Innan 2011). Although gene conversion
reduces the divergence between the paralogous sequences, it
simultaneously increases the variation at the haplotype level
by creating chimeras of the two paralogous sequences
(Fawcett and Innan 2011). It has also been suggested that
gene conversion might be important for buffering crucial
functions in some WGD duplicates (Chapman et al. 2006) or
for maintaining high dosage (Sugino and Innan 2006). In
addition, gene conversion is likely to increase the efficiency
of selection on duplicated genes (Mano and Innan 2008;
17 Significance and Biological Consequences of Polyploidization in Land Plant Evolution
Marais et al. 2010), and it has been reported that the genes
undergoing extensive gene conversion in rice are evolving
faster (diverging faster from their sorghum orthologs) com-
pared to duplicates that are not undergoing gene conversion
(Wang et al. 2009).
17.4 Evolutionary Significance of WGDs
in Angiosperms
Most, if not all eudicots derived from a hexaploid ancestor
(Tang et al. 2008b; Van de Peer et al. 2009b). All
angiosperms might also have a common polyploid ancestry,
as well as all seed plants (Jiao et al. 2011). It is also known
that most vertebrates derived from a common ancestor that
underwent two rounds of WGD (Kasahara 2007). These facts
underscore that polyploids inherently have an enormous
potential for long-term evolutionary success. Here, we will
discuss links between WGDs and two processes that could
have contributed to their evolutionary success: species radi-
ation and the generation of evolutionary novelty. Since hard
evidence for such links is scarce, we will not limit our
discussion to the plant kingdom, but also include evidence
from other organisms, mainly vertebrates and teleost fish.
17.4.1 Genome Duplications and Speciation
Genome duplications have repeatedly been linked to species
radiations, but the evidence in support of this theory is
mostly circumstantial. Examples of putative correlations
between genome duplication and increased diversity are
found among flowering plants and teleost fish.
Soltis et al. studied the relationship between polyploidy
and species richness in angiosperms by comparing the
species richness in clades that are ancient polyploids
with sister clades that are not (Soltis et al. 2009). In
many cases, a strong correlation was found between diver-
sification rates and the occurrence of genome duplications.
Whole-genome duplications have been reported in many
of the most species-rich plant families, including the
Poaceae (>10,000 species), Solanaceae (>3,000 species),
Asteraceae (23,000 species), Fabaceae (19,400 species),
Lauraceae (>2,000 species) and Brassicaceae (3,700
species) (Cui et al. 2006; Fawcett et al. 2009). However,
the phylogenetic position of the WGDs in all of these
families is currently insufficiently resolved to causally
link genome duplications to radiations (Soltis et al. 2009).
The Actinopterygii or ray-finned fish include more than
25,000 species, the vast majority of which belong to the most
derived division, the teleosts. All older, more basal groups of
ray-finned fish, namely the Chondrostei (bichirs, sturgeons
and paddlefish), and the basal neopterygian orders (gars and
285
bowfin), consist of only a few (~44) extant species.
Depending on the source, a fish-specific genome duplication
(3R) is estimated to have occurred in the teleost lineage
between 226 and 350 Mya (Christoffels et al. 2004; Hoegg
et al. 2004; Vandepoele et al. 2004; Hurley et al. 2007).
Phylogenetic analyses indicate that 3R separates the
species-poor early branching ray-finned fish lineages from
the extremely species-rich teleost lineage, suggesting that
3R might be causally related to the teleost radiation. How-
ever, fossil evidence suggests that the major teleost
radiations did not take place until late in the Cretaceous,
more than 150 million years after the youngest 3R age
estimate (Hurley et al. 2007). Thus, there seems to be a
major time gap between 3R and the radiation of the teleosts,
rendering it less likely that genome duplication has been a
causative agent in the radiation process. Could the speciation
potential of 3R have been kept in store for 150 million years?
17.4.2 Reciprocal Gene Loss and Subfunction
Partitioning
Although it has proven difficult to firmly establish causative
links between WGD and speciation, there is at least one
mechanistic process through which gene duplication may
facilitate speciation. WGDs create massive amounts of redun-
dant gene pairs that in the course of evolution return to single-
ton status through duplicate gene loss or pseudogenization. It
can happen that different copies of a duplicated gene pair get
lost in different populations, a process referred to as reciprocal
gene loss (RGL) or divergent resolution. Mating between
individuals from those populations leads to heterozygous F1
progeny with one functional allele at each locus of the
duplicated gene. The F2 offspring will inherit a variable num-
ber of functional alleles, and 1/16 of the progeny will not
inherit any functional alleles. Divergent resolution at 20–30
essential locus pairs could be sufficient to reproductively iso-
late two populations (Werth and Windham 1991; Lynch and
Conery 2000; Semon and Wolfe 2007b). After a genome
duplication, divergent resolution could be operating on
thousands of duplicate pairs simultaneously, rendering it a
very effective speciation mechanism. A similar reproductively
isolating process could occur at the level of duplicate
subfunctionalization (Postlethwait et al. 2004). If a duplicated
gene is multifunctional, different subfunctions could be diver-
gently resolved in different populations and contribute to
reproductive incompatibility. The efficiency of this mecha-
nism would of course depend on the prevalence of subfunctio-
nalization after WGD.
Recently, Bikard et al. found evidence that divergent reso-
lution of duplicated genes can indeed be a source of genetic
incompatibilities (Bikard et al. 2009). These authors studied
crosses between the Col and Cvi accessions of Arabidopsis
286
thaliana and came to the conclusion that a homozygous com-
bination of the Col allele of one particular locus with the Cvi
allele of another unlinked locus led to arrested embryo devel-
opment and seed abortion. In Col, these loci contain a
paralogous pair of histidinol-phosphate aminotransferases,
HPA1 and HPA2. Homozygous HPA1 deletion is embryo
lethal. But in Cvi, the HPA1 ortholog is missing and the
HPA2 ortholog appears to be the functional allele.
The analysis of complete sequences of pre- and post
whole genome duplication species showed that RGL of
duplicated genes after WGD is very common. Scannell
et al. (2006) showed that after the WGD in ascomycetous
yeasts, almost half of the pairs that returned to single-copy
status were divergently resolved, suggesting that RGL could
have been a major factor in post-WGD speciation. No large-
scale studies have been performed so far on plants, but
widespread RGL has also been documented in teleost fish.
Using the post-WGD genome sequences of Tetraodon and
zebrafish, and the pre-genome duplication sequences of
human and chicken as outgroups, it was estimated that
~1,700 WGD-pairs were divergently resolved since the
Tetraodon-zebrafish split (Semon and Wolfe 2007b). The
studies in yeast and fish also suggest that RGL and/or
subfunction partitioning can promote speciation over long
periods of time, even tens to hundreds of millions of years
after a WGD (Scannell et al. 2006; Semon and Wolfe 2007b),
which may help explain how the 3R WGD in teleosts could
have facilitated the teleost radiation 150 million years later.
17.4.3 Dosage Balance Effects and the
Regulatory Spandrel
WGDs leave a very specific signature in the duplicate reten-
tion pattern of certain gene families. In fact, the duplicate
retention behaviour of some gene classes after WGD is the
exact opposite of their behaviour after small-scale
duplications (SSD) (Maere et al. 2005; Freeling 2009).
Many transcriptional and developmental regulators, trans-
porters, signal transducers and complex-forming genes
appear to be preferentially retained after WGD and do not
seem to duplicate easily through SSD. This observation
has been made for WGDs in diverse organisms, from yeast
(Davis and Petrov 2005) and Arabidopsis (Blanc and Wolfe
2004b; Seoighe and Gehring 2004; Maere et al. 2005) to
vertebrates (Blomme et al. 2006; Putnam et al. 2008) includ-
ing teleosts (Blomme et al. 2006; Brunet et al. 2006). The
peculiar reciprocal retention patterns of certain gene classes
after SSD and WGD can be explained by dosage balance
effects (Papp et al. 2003; Birchler et al. 2005). When a
dosage-sensitive gene is duplicated on its own, the effects
of its increased dosage with respect to its interaction partners
or targets would be deleterious, and the duplication would be
J.A. Fawcett et al.
selected against. But if the gene and its interaction partners/
targets are duplicated together, as in WGD, their relative
dosage is preserved. Moreover, if after WGD one of
the duplicates would be lost, this would create a reverse
dosage balance effect, causing dosage sensitive genes to be
selectively retained after WGD.
The consequence of these dosage balance effects is that
post-WGD organisms are endowed with a regulatory ‘span-
drel’ (Gould and Lewontin 1979), a collection of extra
transcription factors, transporters and complexes that may
not immediately be useful but that cannot be purged easily
from the genome. In the long run, these non-adaptively
preserved genes may be co-opted for adaptive innovations.
According to Freeling and Thomas (2006) and Freeling
(2009) the dosage balance-mediated increase of the regu-
latory and complex-forming gene repertoire after WGD
could even cause a predictable drive toward higher complex-
ity, not unlike the meiotic drive.
17.4.4 WGDs and Evolutionary Novelty
It appears that some genome duplications have indeed been
followed by a substantial increase in morphological com-
plexity in the affected organisms. Early WGDs in the angio-
sperm plant lineage have been invoked to explain the rapid
rise and diversification of angiosperms in the Early Creta-
ceous (145–125 Mya) (Crepet 2000; Otto and Whitton 2000;
De Bodt et al. 2005; Otto. 2007; Soltis et al. 2008, 2009; Jiao
et al. 2011), linked to the invention of the closed carpel, the
emergence of double fertilization and the invention of the
flower (Stuessy 2004). Several authors have also suggested a
causal link between the 2R duplications and the emergence
of the vertebrates, (e.g., Ohno 1970; Aburomia et al. 2003;
Holland 2003). The two rounds of genome duplication (2R)
in the vertebrates approximately coincide with the develop-
ment of important morphological innovations, not only in
the endoskeleton but also in the nervous system and sensory
organs, and endocrine, immune and circulatory systems
(Shimeld and Holland 2000; Holland and Chen 2001;
Khaner 2007; Holland et al. 2008; Wagner 2008). Post-3R
teleosts (Stellwag 2004; Heimberg et al. 2008) also devel-
oped some novel morphological features, such as pharyngeal
jaws, an oxygen secretion system to inflate the swimbladder
(Berenbrink et al. 2005), and complex pigment patterning
systems (Mellgren and Johnson 2002; Braasch et al. 2008).
Moreover, there are indications that the oxygen secretion
system originated independently at least four times in
teleosts (Berenbrink et al. 2005), suggesting that these fish
shared the potential to develop such a system whereas fish
that did not undergo the 3R duplication did not.
It is unclear to what extent genome duplications are
involved in the actual invention of fundamental innovations.
17 Significance and Biological Consequences of Polyploidization in Land Plant Evolution
There are indications that genome duplications may play a
more subtle role instead, in refining and elaborating
innovations rather than inventing them. In many organisms,
fundamental innovations have been followed by the emer-
gence of many variants or elaborations. The invention of the
flower for instance was followed by the development of a
huge variety of floral forms, specialized pollination
syndromes and fruits. Many of the morphological
innovations in vertebrates also derive from a single key
innovation: the neural crest, a migratory cell population
that gives rise to numerous differentiated cell types (Shimeld
and Holland 2000; Holland et al. 2008). The evolutionary
success of the vertebrates is as much related to the develop-
ment of various neural crest elaborations as to the invention
of the neural crest itself. The complex pigment patterning
systems of post-3R teleost fish for instance also find their
origin in a further expansion of the neural crest-derived cell
type repertoire (Mellgren and Johnson 2002; Braasch et al.
2008).
It is likely that regulatory spandrels formed by WGD may
have contributed to the elaboration, if not invention, of the
key morphological innovations in angiosperms and
vertebrates. In angiosperms, many regulatory gene families
that exhibit WGD-mediated expansion, e.g., the Aux/IAA
auxin response regulators (Remington et al. 2004) and some
MADS-box transcription factor subfamilies (Zahn et al.
2005; Veron et al. 2007), are directly involved in the devel-
opment of morphological features that are considered angio-
sperm inventions and derivatives thereof. The same is true
for many of the expanded regulatory gene families in
vertebrates, such as homeobox genes (Holland and Garcia-
Fernandez 1996; Holland 2003; Holland et al. 2008) and
neural crest specifier genes (Holland et al. 2008).
There is an interesting parallel to be drawn between the
origin of novel gene functions through gene duplication and
the origin of innovations through genome duplication. Evo-
lution after single gene duplication has been studied for
decades, and many models have been developed to explain
how gene duplicates can develop new functions. The classi-
cal neofunctionalization model of Ohno (1970) starts from
the premise that a duplicate gene is free from selection and
that, by genetic drift, it might discover a new beneficial
function. It has since become clear that the vast majority of
such neutrally evolving duplicates will be lost long before
they can ever neofunctionalize. Alternatively, duplicates of
multifunctional genes may be preserved through subfunctio-
nalization, i.e., partitioning of the different subfunctions of a
gene over the different copies (Force et al. 1999). But
subfunctionalization in itself does not lead to novelty.
More recently, two models have been proposed that combine
aspects of sub- and neofunctionalization, namely the
‘Escape from Adaptive Conflict’ (EAC) model (Hittinger
and Carroll 2007; Des Marais and Rausher 2008) and the
287
‘Innovation, Amplification, Divergence’ (IAD) model
(Hendrickson et al. 2002; Francino 2005; Bergthorsson
et al. 2007). The central idea of these models is that second-
ary functions of an ancestral gene can get co-opted to a
primary role in one of the gene duplicates (Conant and
Wolfe 2008). In other words, the ‘new’ function in the
duplicated gene does not arise de novo but is already present
in a seminal form in the ancestral gene. But the primary and
secondary functions in the ancestral gene may be subject to
pleiotropic constraints, precluding optimization or elabora-
tion of both functions simultaneously. Gene duplication
offers the opportunity to escape from such adaptive conflicts,
allowing natural selection to optimize the primary and sec-
ondary function independently in different copies.
A similar mechanism might be operating on the level of
functional innovation through genome duplication. Analo-
gous to adaptive conflicts in single genes being resolved
through gene duplication, adaptive conflicts in gene
networks could be resolved through genome duplication.
As in the EAC and IAD models of evolution after gene
duplication, genome duplications may be instrumental in
lifting pleiotropic constraints in molecular networks and
perfecting primitive versions of network-level inventions,
rather than facilitating innovation de novo.
17.4.5 WGDs Increase Evolutionary Potential,
and May Do So for a Long Time
What WGDs essentially do is increase the evolutionary
potential of an organism. But this potential is not necessarily
fulfilled soon (or ever (Semon and Wolfe 2007a)).
Circumstances like the availability of niche space are
equally important in determining whether or not a post-
WGD organism can advantageously use its genomic span-
drel. And even under the right conditions, expressing the
evolutionary potential of a lineage likely takes time and
proceeds through a series of intermediate forms that are
later outcompeted by more derived relatives (Knoll and
Carroll 1999; Feild and Arens 2007). Indeed, Donoghue
and Purnell (2005) have observed that there are no bursts
in morphological innovation in post-WGD clades when tak-
ing into account extinct lineages. Although a genome dupli-
cation can be considered a saltational event in terms of
genome evolution, the immediate phenotypical or morpho-
logical impact is usually limited. Post-WGD organisms may
be considered Goldschmidtian ‘hopeful monsters’ in the
genomic sense, but most often not in the phenotypic sense
(Goldschmidt 1940).
Given the importance of opportunity and time in devel-
oping evolutionary innovations, a crucial feature of WGDs is
that their evolutionary potential can be preserved over long
periods of time. Dosage balance effects and functional
288
divergence mechanisms such as subfunctionalization can in
principle cause indefinite preservation of duplicate genes,
and in particular the regulatory spandrel, in the absence of
positive selection. A recent study on the functional diver-
gence of Scn4aa and Scn4ab, a duplicated pair of sodium
channels remaining from the teleost-specific genome dupli-
cation, confirms that such duplicated genes can neofun-
ctionalize and contribute to morphological innovations
more than 100 million years after they were duplicated
(Arnegard et al. 2010). The authors investigated whether
neofunctionalization of Scn4aa could be directly linked to
the origin of adult myogenic electric organs, which hap-
pened 100 million years after 3R and on two separate
occasions, in African mormyroid and South American
gymnotiform fishes. They found that in both lineages,
Scn4aa has been co-opted for exclusive expression in
myogenic electric organs, and that coinciding with gene
co-option, functionally important motifs of Scn4aa evolved
under positive selection, suggesting that Scn4aa has been
instrumental in the evolution of myogenic electric organs.
17.4.6 Opportunities, Niches and Mass
Extinctions
Compared to the high frequency of polyploidization in mod-
ern plants (in the order of 1 in 105 individuals) (Ramsey and
Schemske 1998), the number of preserved ancient genome
duplications in plants is low. This pattern is even more
conspicuous in vertebrates and yeast (Van de Peer et al.
2009a). Thus, despite the success of some WGD-derived
lineages such as the angiosperms, vertebrates, or teleost
fish, it appears that genome duplications are seldom success-
ful on long evolutionary timescales. The diversification of a
group of species likely depends largely on the ecological
opportunities. Even if polyploids possess the potential for
the diversification of species and evolutionary novelty, such
potential is unlikely to be realized unless an ecological
opportunity presents itself. In normal, stable ecosystems,
newly formed polyploids are probably not able to compete
with the highly adapted occupants of existing niches, includ-
ing their diploid ancestors (Conant and Wolfe 2007; Feild
and Arens 2007; Otto 2007; Hegarty and Hiscock 2008). The
number of available niches might be a limiting factor for
polyploid lineages to further diversify into species-rich
groups. Then, how might the various polyploid ancestors
of the extant plant species have managed to survive and
diversify? Although it is difficult to know the environmental
conditions in which ancient polyploids originated, recent
evidence indicates that large-scale ecological catastrophes
might have been instrumental in setting the stage for novel or
young polyploid lineages to diversify (Fawcett et al. 2009;
Van de Peer et al. 2009a). Fawcett et al. (2009) found that
J.A. Fawcett et al.
many angiosperm plant lineages independently underwent a
genome duplication around the Cretaceous-Tertiary (K-T)
boundary, ~65 Mya. The K-T event is the most recent large-
scale mass extinction of animal and plant species, catalyzed
at least in part by one or more catastrophic events such as an
asteroid impact on the Yucatan peninsula and increased
volcanic activity in India. The plant lineages that underwent
WGD around the K-T boundary gave rise to some of the
most species-rich angiosperm families such as the Poaceae,
Solanaceae, Asteraceae, and Fabaceae. The proposed
catalyzing function of mass extinctions in WGD establish-
ment combined with the fact that WGDs increase the
evolutionary potential of a lineage suggest somewhat para-
doxically that mass extinctions may ultimately lead to more
complex plants.
17.5 Conclusions and Perspectives
We are now witnessing a dramatic increase in the availabil-
ity of whole-genome sequences in plants. As the whole-
genome sequence of A. thaliana has been instrumental in
efforts to understand the biology of A. thaliana, a large
number of plant genome sequences is likely to help us better
understand the molecular basis of the morphological diver-
sity encoded within the different plant genomes. The current
morphological diversity is the consequence of a long history
of evolutionary and ecological processes, and as we have
discussed in this chapter, the evolution of plants, or at least
flowering plants, is also the evolution of polyploids. We now
know that most eudicots, and possibly all angiosperms are
derived from polyploids, and that many of the most species-
rich families are also derived from independently formed
polyploids (Tang et al. 2008b; Fawcett et al. 2009; Soltis
et al. 2009; Van de Peer et al. 2009a). The evolutionary
success of various polyploid lineages was probably made
possible by their potential for successive rounds of specia-
tion by reciprocal gene loss, and their potential to evolve
new functions or morphological features due to the duplica-
tion of the entire gene content (in particular the regulatory
spandrel) and genetic network (Edger and Pires 2009;
Kejnovsky et al. 2009; Van de Peer et al. 2009a). However,
concrete examples of such processes are still scarce. We
know little about how reciprocal gene loss following
polyploidization actually contributed to the species diversi-
fication of the various angiosperm lineages. This probably
requires a considerable number of genome sequences of
species that diverged from each other after polyploidization,
which are not available now, but might be in the future. We
also do not know much about how the different polyploid-
derived lineages achieved specific adaptations and evolved
different molecular functions and morphological features.
17 Significance and Biological Consequences of Polyploidization in Land Plant Evolution
This requires further understanding of the biology and vari-
ous molecular aspects of different plant species apart from a
few model species such as Arabidopsis, with the aid of the
genomic information becoming available. It is also impor-
tant to note that most of our knowledge regarding WGDs in
plants comes from angiosperms, with the exception of the
moss Physcomitrella patens (Rensing et al. 2007; 2013 this
volume) and the fern Ceratopteris richardii (see Barker
2013, this volume). Examining the impact of WGDs in
non-angiosperm plant species will be necessary in order to
understand the full extent of the significance of WGDs in the
evolution of plants.
Acknowledgments SM is a fellow of the Fund for Scientific
Research-Flanders. JAF is a JSPS (Japan Society for the Promotion of
Science) postdoctoral fellow.
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 Evolutionary Importance of Generative
Polyploidy for Genome Evolution of 18
Haploid-Dominant Land Plants

Stefan A. Rensing, Anna K. Beike, and Daniel Lang

Contents 18.1 Introduction

18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
18.2 History Repeating: Artificial and Natural Polyploidy in
Generative polyploidy is a widespread phenomenon among
the Funariaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 land plants and has been pivotal to the evolution of land
plant genomes (Soltis and Soltis 2009; Van de Peer et al.
18.3 Natural Polyploidization and Hybridization Among the
Funariaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 2009, Fawcett et al. 2013, this volume). These large-scale or
whole genome duplication events (WGD) are widely
18.4 Gene Retention and Gene Family Evolution . . . . . . . . . . . 300
recognized as driving forces behind diversification and spe-
18.5 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 ciation of plant lineages and have often been hypothesized as
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 important factors for the evolution of organismal complexity
(Crow and Wagner 2006; Van de Peer et al. 2009). Initial
evidence for the latter hypothesis was recently provided by
phylogenetic comparative genomics revealing a correlation
of WGD with morphological complexity (Lang et al. 2010).
Autopolyploidization, i.e., the doubling of the genome
without hybridization, might represent a short-term evolution-
ary advantage during periods of environmental change that
force range shifts (Parisod et al. 2010). Allopolyploidization,
i.e., the doubling of the genome in association with
hybridization of genetically different chromosome sets, is
also regarded as enabling an increased potential for sub- and
neofunctionalization of genes and thus speciation (Soltis and
Soltis 2009). Allopolyploidization has also been hypothesized
to be correlated with periods of environmental upheaval
(Fawcett et al. 2009, 2013).
Key to our understanding of the evolutionary importance
of WGD events and especially their effects on diversification
is the fate of the duplicated chromosomal regions. The
diversifying effects of this post-duplication phase encompass
large-scale phenomena like subsequent diploidization (or
haploidization, in the case of haploid-dominant plants), chro-
mosome loss and rearrangements, as well as small-scale,
adaptive processes related to the functionalization and diver-
sification of duplicated gene paralogs; which in the case of
WGD are termed paleologs or homoeologs. There are various
S.A. Rensing (*)
models describing the fate of paralogs, covered comprehen-
Faculty of Biology, University of Freiburg, Sch€anzle str. 1, Freiburg sively by, for example, Innan and Kondrashov (2010).
79104, Germany
e-mail: stefan.rensing@biologie.uni-freiburg.de

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 295

DOI 10.1007/978-3-7091-1160-4_18, # Springer-Verlag Wien 2013
296
It is presumed that most duplicated segments are lost
quickly during the early phases of diploidization (Ma and
Gustafson 2005). Even if duplicated segments survive the
early post-duplication phase and the retained homoeologs are
becoming fixed in the population, the most common fate is
death by gradual pseudogenization and non-functionalization,
except if a selective advantage is introduced. Therefore, central
to our understanding of the importance of generative poly-
ploidy is why certain homoeologs are retained and which
models can be applied to describe their distinct evolutionary
paths which fall somewhere between the extremes of diversifi-
cation leading to the acquisition of new gene functions and
redundancy which is thought to be important for gene dosage
(Innan and Kondrashov 2010).
Most of the evolutionary models describing the post-
duplication fates of paralogous gene copies have been
developed and applied to explain gene duplication in
diploid-dominant species, like seed plants that exhibit a
heterophasic life cycle with a dominant diploid sporophytic
generation. Indeed they are the major focus of the discussion
of generative polyploidy in the chapter by Fawcett et al.
(2013), this volume. Here we focus on aspects and effects of
polyploidization in haploid-dominant plants, like bryophytes,
which have a dominant haploid gametophyte and a reduced
diploid sporophytic generation. What role does polyploi-
dization play in the evolution of these organisms? Can we
find evidence of ancient and recent polyploidization events in
the genomes of extant bryophytes? What is the importance of
auto- versus allopolyploidy? What is the extent of the
paranome, i.e., the genomic fraction of paralogs, within a
bryophyte genome? What is the prevalent model for
functionalization of paralogs and which kind of functional
classes of genes have been retained preferentially?
Bryophytes form a paraphyletic group at the base of the
land plant tree of life and comprise liverworts, mosses and
hornworts—listed in order of currently assumed divergence
from the vascular plant lineage (Qiu et al. 2006; see also Soltis
and Soltis 2013, this volume). Although the fossil record of
early land plants is scarce, there are fossils indicating that
early land plants belonged to the liverwort-lineage (Kenrick
and Crane 1997; Wellman et al. 2003). The last common
ancestor of vascular (diploid-dominant) and non-vascular
(haploid-dominant) land plants was recently dated to have
lived about 500 million years ago (Mya) (Lang et al. 2010).
As mentioned above, this deep divergence has led to a radi-
cally different approach to life on land compared with seed
plants, including poikilohydry (i.e., the change of cellular
water content in response to water availability in the environ-
ment) and the dominance of the gametophytic generation,
which results in a haploid state of the genome for the domi-
nant part of the heterophasic life cycle.
It is tempting to speculate that WGD (comprising auto-
and allopolyploidizations) represent an even greater poten-
tial evolutionary advantage in haploid-dominant organisms
S.A. Rensing et al.
(like bryophytes) than in those that spend most of their life
cycle in the diploid phase (like seed plants, monilophytes
and lycophytes). The presence of an additional copy of each
gene and chromosome after a WGD might render a bryo-
phyte more robust against somatic mutations eventually
affecting the germ line. In addition, it would have the same
potential to evolve new functions as in diploid-dominant
organisms. Indeed it has been shown that allopolyploid
diploids of the moss Sphagnum subsecundum do exhibit
greater genetic diversity and linkage disequilibrium than
haploids (Shaw et al. 2008).
The estimated frequency of polyploid species differs
between the three bryophyte phyla and between authors. For
example, Wyatt et al. (1988) estimated that about 79% of
mosses, 11% of liverworts and 2% of hornworts are naturally
occurring polyploids. Examples include the liverwort Pellia
borealis (Odrzykoski et al. 1996; Orzechowska et al. 2010),
and various moss genera including Sphagnum (Shaw et al.
2008; Karlin et al. 2009; Ricca and Shaw 2010), Plagiomnium
(Wyatt et al. 1988, 1992) and Physcomitrella/Physcomitrium.
The proposed widespread existence of naturally occurring
generative polyploidy within mosses (reviewed by Natcheva
and Cronberg 2004), as well as the potential differences
compared with diploid-dominant plants reviewed in Husband
et al. (2013, this volume), make mosses a particularly inter-
esting subject to study the influence and implications of WGD
for the evolution and biology of haploid-dominant organisms.
Heterosis effects of WGD, especially with regard to body
size, might explain the sudden increase in size of mosses
observed in extant species compared with those embedded in
Eocene (45 million year old) amber (Frahm 2010). Indeed,
the estimated timing (30–60 Mya) of the WGD event
in the model moss Physcomitrella patens (Hedw.) Bruch &
Schimp. (Rensing et al. 2007) falls in the period of the mass
extinction events of the “Grand Coupure” at the end of the
Eocene (33 Mya) and of the Cretaceous-Tertiary (K-T)
boundary (65 Mya).
Physcomitrella patens has been developed as a model
organism over the last two decades, and a well-developed
molecular toolbox including efficient gene targeting (Frank
et al. 2005; Cove et al. 2009) is now available together with
sequenced nuclear and organellar genomes (Sugiura et al.
2003; Terasawa et al. 2007; Rensing et al. 2008). This makes
P. patens, and the family Funariaceae to which it belongs,
ideal candidates to study and demonstrate the effects of
polyploidization on haploid genomes.
18.2 History Repeating: Artificial and Natural
Polyploidy in the Funariaceae
In the early twentieth century the moss family Funariaceae
was extensively studied by von Wettstein in his ground-
breaking work on its genetics and the appearance of both
18 Evolutionary Importance of Generative Polyploidy for Genome Evolution of Haploid-Dominant Land Plants 297

Physcomitrium eurystomum
Physcomitrium sphaericum
Physcomitrium turbinatum
Physcomitrium immersum

Physcomitrium pyriforme

Entosthodon fascicularis
Physcomitrella patens

Funaria hygrometrica
Funaria mediterranea
Funaria cricetorum
Physcomitrella patens X natural hybrids
Physcomitrium sphaericum X experimental hybrids
Physcomitrium turbinatum
Physcomitrium immersum Fertility of spores
Physcomitrium eurystomum many
Physcomitrium pyriforme some
Funaria hygrometrica not fertile
Funaria mediterranea very few found
Funaria cricetorum not reported
Entosthodon fascicularis
Sporophyte morphology
intermediate
mother or mother-like
intermediate with tendency to mother-like
not reported

Fig. 18.1 Matrix of natural and experimental hybrid occurrence in the Funariaceae

natural and experimental hybrids (Wettstein 1924, 1932). gametes are able to fertilize eggs from a range of other
His results are shown in Fig. 18.1 and demonstrate that Funariaceae, they are not able to pass on the elaborate
hybridization is quite common within this family. If hybrids sporophytes to their hybrid offspring. It might well be that
are fertile and thus able to develop sporophytes, these are aneuploidization, as has been hypothesized for P. patens
usually of intermediate or mother-like morphology and the (Rensing et al. 2007; Beike and Rensing 2010), is the basis
spores are sometimes few and often not fertile (Fig. 18.1). of this behaviour.
While P. patens seems to represent a good mother line, The hybrid lines mentioned above are usually homoploid
Funaria hygrometrica is a good father line. Interestingly, hybrids (i.e., lacking a WGD) and as they are haploid no allele
these two species occupy the opposite ends on the complex- can cover for essential functions if the mating partner’s genome
ity scale of sporophyte morphology; while F. hygrometrica is altered in that regard. Consequently, a lot of the hybrid
features an elaborate sporophyte with a long seta (the stalk offspring are not expected to survive due to severe incompati-
on which the spore capsule rests) and operculum (the pre- bility or will be sterile. However, as mentioned earlier,
formed breaking point for the release of spores), P. patens Physcomitrella is a paleopolyploid (Rensing et al. 2007) and
harbours a highly reduced sporophyte without operculum molecular dating of the WGD event implies ancient polyploi-
that, due to the extreme reduction of the seta, is embedded dization in the ancestor of the Funariaceae (see below). This
into the gametophores that gave rise to it. The reduced may explain, in part, why natural hybrids like Physcomitrium
sporophyte of P. patens was recently proposed to be a collenchymatum or Physcomitrium eurystomum are able to
secondary reduction of a more complex, potentially survive. Crosses of P. patens isolates, that may very well
operculum-bearing sporophyte in the Funariaceae ancestor represent cryptic species, usually display a slightly aberrant
(McDaniel et al. 2010). Studies have shown that the sporophyte morphology (see Fig. 18.2d and 18.2e, compared
chromosomes of P. patens are apparently able to pair and with selfing, Fig. 18.2a), but do produce fertile spores, whereas
recombine with those from several Funariaceae species and crosses with, for example, Physcomitrium, if they occur at all,
usually force the reduced phenotype onto the hybrid off- may result in intermediate sporophyte morphology, as men-
spring (Fig. 18.2). In contrast, while the F. hygrometrica tioned above (Fig. 18.2b). It should be noted that it is not only
298
Fig. 18.2 Sporophytes resulting from (a) selfing of Physcomitrella patens and (b) P. patens crossed with Physcomitrium sphaericum and (c and f)
selfed again. (d) and (e) show sporophytes from crosses between different isolates of P. patens
the hybrid sporophytes arising directly from crossings that
display the intermediate morphology, the same is true also for
the following generation, i.e., sporophytes eventually formed
upon germination of the spores and selfing of the resulting
gametangia (Fig. 18.2c, f).
Artificial or experimentally induced autopolyploidization
is also frequently observed after transfection of P. patens
protoplasts (12%; Schween et al. 2005b). Among
transformants the polyploid, mostly diploid, plants cannot
be distinguished from homoploid, haploid plants by morpho-
logical features alone (Schween et al. 2005a).
18.3 Natural Polyploidization and
Hybridization Among the Funariaceae
The existence of natural polyploids derived from unreduced
spores (diplospory), by reprogramming of vegetative sporo-
phytic cells (e.g., after wounding; apospory) or fusion of
sporocytes prior to meiosis (syndiplospory) is known for
individual moss plants (Crawford et al. 2009). Also, genera-
tive polyploidy at the population or species level has been
known for a long time, as outlined above. Nevertheless, the
S.A. Rensing et al.
question as to which of the two forms of genome doubling is
most prevalent among bryophytes has been an issue of
debate. In contrast to allopolyploidy, von Wettstein consid-
ered autopolyploidy to be of little or no evolutionary impor-
tance for mosses due to the abnormality and cytological
instability of the resulting moss strains (reviewed in English
by M€untzing 1936). In contrast, in the 1970s it was assumed
(Longton 1976; Smith 1979) that most polyploid bryophytes
were autopolyploids (with a few exceptions) and thus
hybridization followed by genome duplication (i.e., allo-
polyploidy) was of little significance.
Recent molecular studies on polyploid bryophytes show
that allopolyploidy is the rule rather than the exception, and
is common in both mosses and liverworts (Natcheva and
Cronberg 2004). After hybridization, allopolyploidization
may occur in three ways: (1) apospory (i.e., regeneration of
diploid gametophytes from the tissues of the hybrid sporo-
phyte), (2) diplospory, or (3) syndiplospory.
Different chromosome counts have been reported for
Funariaceae with the most common numbers being 14, 21
and 28. In P. patens the chromosome number per haploid cell
varies from n ¼ 14, n ¼ 16 as reported by von Wettstein
(1924) and Engel (1968) to n ¼ 27 reported by Bryan
18 Evolutionary Importance of Generative Polyploidy for Genome Evolution of Haploid-Dominant Land Plants
(1957) and Reski et al. (1994) with the latter count being
recorded for the isolate “Gransden 2004” used for genome
sequencing (Rensing et al. 2008). Among true mosses
(Bryopsida), the base number of chromosomes is considered
to be four, five, six and seven (Frahm 2001). Considering a
base number of seven chromosomes among Funariaceae, a
model for the genome evolution of P. patens can be
constructed based on two whole genome duplication events.
Starting with the ancient hybridization of two (male and
female) parental chromosome sets of n ¼ 7, the resulting
allopolyploidization and subsequent haploidization (i.e., a
loss of complete redundancy by paralog decay and sub- and
neofunctionalization) may have led to a haploid, mon- and
synoicous species with a chromosome number of n ¼ 14.
Another (auto- or allo-) polyploidization and haploidization,
followed by a putative aneuploidization, would result in the
extant haploid P. patens strain with 27 chromosomes
(Rensing et al. 2007; see also Fig. 3 in Rensing et al. 2009
for illustration). Thus, the last common ancestor of
Funariaceae potentially became hermaphroditic through
hybridization and polyploidization (Rensing et al. 2007).
Evidence that this might reflect a more general trend among
mosses comes from a recent phylogenetic comparative study
indicating that polyploid mosses were more likely to be her-
maphrodite, thus suggesting that changes in chromosome num-
ber through polyploidy could cause changes in the sexual
system (Crawford et al. 2009). The selective advantage of
hybridization and polyploidization depends on successful
reproduction and transmission of genetic information. The
evolutionary benefit of a syn- and monoecious moss species
over a dioicous species might be the easier way of sexual
reproduction through selfing, as male and female gametangia
are located on the same plant, and even on the same gameto-
phore. However, inbreeding depression can be a considerable
selective disadvantage of monoecy, even if out-crossing among
monoecious bryophytes occurs in nature as already described
for the synoicous polyploid moss Plagiomnium medium (Wyatt
et al. 1988, 1992). Interestingly, sporophytic inbreeding depres-
sion was noted in the dioicous species Ceratodon purp-
ureus, but not in the monoecious, self-fertilizing Funaria
hygrometrica. Indeed, the low rate of inbreeding depression
observed in selfing F. hygrometrica, based on capsule and seta
length, spore number or capsule mass, may help in maintaining
the evolutionary stability of hermaphroditic populations
(Taylor et al. 2007). In addition, among bryophytes, the ability
and high frequency of vegetative reproduction via fragmenta-
tion of gametophytic tissue or gemmae should be considered in
this context. A successful vegetative reproduction system can
diminish the potentially negative consequences of sexual isola-
tion through polyploidization.
P. patens is a paleopolyploid moss that underwent at least
one WGD (Rensing et al. 2007). This ancient polyploidization
was identified from an analysis of 2,907 paralogous genes out
299
of 24,845 transcriptomic unigenes. Based on the synonymous
substitution rates of homoeologs, the divergence of the
paralogous gene copies was dated to 30–60 Mya ago. An
average of 45 Mya can therefore be expected for the date of
WGD in P. patens. According to phylogenetic analyses, the
age of the subclass Funariidae has been estimated to be approx-
imately 172 Mya (Newton et al. 2007). Therefore, the detected
genome duplication during the Eocene (~45 Mya) most likely
occurred after diversification within Funariidae. Although the
age of Funariaceae is still unclear, molecular analysis of a
conserved domain of MADS-box transcription factors in
F. hygrometrica, Funariella curviseta, P. eurystomum and
P. patens hints at a shared polyploidization history within this
family (manuscript in preparation; Zobell et al. (2010)). The
analysed species share the same MADS-box transcription
factors, which are known to be preferentially retained after
duplication events (Veron et al. 2007).
For some time now, bryologists have discussed monoicy as
a derived trait over dioicy (Ando 1980). For dioicous mosses
reproductive success is strongly dependent on the cytotype
frequencies in the population (Crawford et al. 2009), therefore
asexual reproduction might be predicted to be more frequent
among dioicous than monoecious mosses. In terms of the
distribution of sexual reproductive systems among
Funariidae, both Encalyptales and Timmiales contain monoe-
cious and dioicous species, and among the Funariales only the
family Funariaceae is entirely monoecious. The extension of
the phylogenetic analysis of homoeolog-containing gene
families, like the MADS-box transcription factors mentioned
above, to include additional representatives of the Funariidae
will provide further insight concerning the role of WGD
events in the establishment of monoicy in mosses.
The genome duplication during the Eocene provides no
explanation for the range of chromosome numbers encoun-
tered in extant Funariaceae. However, since several
Funariaceae show no effective intergeneric breeding barrier,
and since some genera, especially Funaria, Physcomitrium
and Physcomitrella, grow in comparable habitats in close
proximity, a high number of hybrid species can be expected
in nature. Thus speciation through hybridization may be
caused by reproductive isolation due to allopolyploidy and
this may contribute to the range of chromosome numbers
observed. It is noted that although some hybrid species from
different moss families have been observed in nature, the
evolutionary significance and the frequency of hybridization
among bryophytes is considered to be widely underestimated
at present (Natcheva and Cronberg 2004).
Natural hybrids among Funariaceae have frequently been
reported over the last two centuries (Britton 1895; Andrews
1918, 1942; Pettet 1964), and outlined above. Recently,
the hybrid origin of Physcomitrium eurystomum and
Physcomitrium collenchymatum was verified based on molec-
ular data and genealogical analyses of six loci, including the
300
ribosomal internal transcribed spacer (ITS), one plastid marker
(atpB-rbcL) and four protein coding loci. Furthermore, out of a
taxon set comprising Funariaceae species from geographically
widespread populations in both the Northern and Southern
Hemisphere, other Physcomitrium species also showed a clear
signature of hybridization (i.e., there were discrepancies in the
phylogenetic trees of the six chosen loci). Moreover, the ampli-
fication of a highly conserved nuclear single copy gene (BRK1)
from a comparable Funariaceae taxon set, including multiple
isolates of four Physcomitrium species, has underlined the
hybrid origin of P. eurystomum and P. collenchymatum.
BRK1 is present as multiple paralogs in the respective species,
which are recognized through sequence polymorphisms in
terms of multiple peaks in the direct sequencing products.
Both distinct parental homologs were identified by cloning
the PCR products and then sequencing several clones per
species isolate. In addition, different isolates of P. pyriforme
(one of the parental lines giving rise to P. eurystomum and
P. collenchymatum) were shown to contain multiple paralogs
of the single copy gene BRK1. Flow cytometric measurements
revealed all those isolates as recent allopolyploids
(neopolyploids), since they have a larger genome size com-
pared to other Funariaceae (manuscript in preparation). Chro-
mosome counts of n ¼ 9 to n ¼ 72 for P. pyriforme, and
n ¼ 9 to n ¼ 54 for P. eurystomum (Fritsch 1991) also suggest
a high frequency of putative hybrid species generation in the
genus Physcomitrium. In contrast, there is so far no evidence
for a hybrid origin or recent allopolyploidy within
Physcomitrella. The genomic DNA content of the different
isolates is comparable to the well characterized Gransden iso-
late and the direct sequencing of the single copy gene BRK1
was unambiguous (manuscript in preparation).
Initially, Physcomitrella was subdivided into three spe-
cies (Tan 1978). P. readeri from USA, Australia and Japan
(Ochi 1968), P. magdalenae from Rwanda and the Demo-
cratic Republic of Congo, Africa (Sloover 1975; Mueller
1995) and P. patens with a wide distribution in the Northern
Hemisphere. Based on variable, but overlapping, phenotypic
and key taxonomic characteristics of these species, the taxon
Physcomitrella was later subdivided into four subspecies:
P. patens subsp. patens, P. patens subsp. magdalenae de
Sloover, P. patens subsp. readeri C. Muell., and P. patens
subsp. californica Crum & Anderson (Tan 1979). Current
molecular data however cannot confirm this latter classifica-
tion. Instead, phylogenetic analyses of different loci, includ-
ing plastid, ribosomal and nuclear marker genes, show three
distinct lines of Physcomitrella which are clearly separated
from each other. These data therefore suggest that (1) the
genus is polyphyletic and arose at least three times from
distinct ancestors within Funariaceae (more or less mirroring
the older classification scheme) and (2) the secondary reduc-
tion of the sporophyte has occurred independently in each
lineage. With regard to these molecular and phylogenetic
S.A. Rensing et al.
analyses, a revised classification for the Physcomitrium/
Physcomitrella species complex, following the initial taxon-
omy (Tan 1978), might be necessary.
18.4 Gene Retention and Gene Family
Evolution
Previous studies of WGD events in P. patens were based on
transcriptome representations (Rensing et al. 2007) and in
order to avoid false-positives due to redundant representation
of transcripts (by means of several unigenes per locus), nearly
identical sequences were removed from such analyses.
Repeating the analysis of Rensing et al. (2007) but based on
the most recent genomic data gives the results shown in
Fig. 18.3 (Ks plot, synonymous substitutions per site for
paralog clusters). The first bin (synonymous substitutions
per site between 0.0 and 0.1) contains a higher number of
paralogs as previously reported (Rensing et al. 2007), i.e.,
~40%, exceeding the estimate based on transcriptome
data. The WGD (secondary peak) comprises another ~40%
of the genes. The latter homoeologs show significant
sequence variation and have probably undergone sub- and
neofunctionalization (Ohno 1970; Wendel 2000). Mutant
analysis of closely related paralogs, usually retained after
WGD, has shown in several cases that the proteins are often
partly functionally redundant, so that they all need to be
down-regulated in order to exhibit a drastic aberrant pheno-
type, yet they differ, for example, in their expression pattern.
Examples of such P. patens gene families are rad51
(Markmann-Mulisch et al. 2002, 2007), lhcsr (Alboresi et al.
2010), rsl (Menand et al. 2007) and ftsZ (Martin et al. 2009a,
b) (i.e., knockout mutants of the homoeologs all show par-
tially redundant or additive phenotypes, yet differential
expression levels or domains). These observations indicate
that both, gene dosage-dependent redundancy and gene
subfunctionalization have occurred. More detailed phyloge-
netic and experimental analysis is necessary to ascertain the
contribution of these different types of evolution following
polyploidy.
Paralogs from the first bin of Ks plots are usually consid-
ered to be the result of local duplication events, such as
segmental duplications (Lynch and Conery 2000; Blanc
and Wolfe 2004a). Indeed, analysis of the P. patens genome
has demonstrated that ~1% of the genes are located in
tandem arrays, i.e., (often identical) paralogs are in close
proximity to each other (Rensing et al. 2008). Currently, we
do not know whether such tandemly arrayed genes are the
result of recent duplications (which are prone to subsequent
paralog loss) or whether they are kept alike by concerted
evolution/gene conversion (Wendel 2000; Wang et al.
2007). Even more intriguing is the high proportion of
genes that are nearly identical but are not tandemly arranged
18 Evolutionary Importance of Generative Polyploidy for Genome Evolution of Haploid-Dominant Land Plants
Fig. 18.3 Ks plot of Physcomitrella patens paralogs. The plot was generated as previously described (Rensing et al. 2007) but is based on the most
recent gene predictions and does not feature a cutoff criterion for nearly identical sequences
(hypothesized from the large first bin, Fig. 18.3). Whether
this is due to the still fragmented nature of the genome
assembly or reveals a complex pattern of intra- and inter-
chromosomal gene conversion has yet to be determined.
This pattern might be related to the moss’ high rate of
DNA repair by homologous recombination and be indicative
of concerted evolution of these loci via gene conversion to
allow the haploid organism to maintain ‘pseudoalleles’ of
dosage-sensitive or highly expressed genes (Lang et al.
2008). Additional evidence that gene conversion is active
in P. patens comes from the analysis of the ftsZ gene family,
where conversion tracts have been shown to maintain func-
tional tubulin/GTPase domains among ftsZ homoeologs
(Martin et al. 2009b), mirroring the pattern predicted on a
larger scale for rice paralogs (Wang et al. 2007).
Enrichment analysis of functional annotations of both
paleologs (Rensing et al. 2007) and pseudoalleles (Lang
2008) in P. patens revealed an overrepresentation of gene
products involved in metabolic processes. This is in clear
contrast to what was found previously for seed plant
genomes, where the genes involved in signal transduction
and transcriptional regulation were the ones that were found
to be preferentially retained, e.g., in Arabidopsis thaliana
(Seoighe and Gehring 2004; Blanc and Wolfe 2004b). This
striking difference can at least partly be interpreted in light of
Funariaceae ecology. Species belonging to the family are
typically short-lived and annual (maximally biennial). This
contrasts with the perennial life style that is common in many
301
other moss families. Based on their adopted life strategies,
Funariaceae are pioneer plants, classified as fugitives and
annual shuttle species, implying they have adapted to open,
unshaded and unstable habitats where survival depends on
their ability to pass quickly through their life cycle when the
conditions are optimal and competition from other species is
low. Thus, the observed functional enrichment of metabolic
genes might reflect a specific adaptation, ensuring the correct
gene dosage important to their particular ecological niche. It
might also reflect, for example, the poikilohydric life style of
bryophytes in general. Poikilohydry enables bryophytes to
resuscitate and restart their entire metabolism almost
instantly after suspending it to endure various environmental
stresses (e.g., heat, drought, cold, radiation). Indeed, the high
abundance of metabolic genes has been found not only in
P. patens but also in the rehydration transcriptome of Tortula
ruralis (Oliver et al. 2004). Furthermore, genes in metabolic
pathways have recently been found to constitute important
fractions of retained paralogs in other species (the protozoon
Paramecium, and yeast) as well (Gout et al. 2009; van Hoek
and Hogeweg 2009).
18.5 Outlook
Physcomitrella patens was initially selected for genome
sequencing because it was already well established as a
model organism with the full molecular and genetic toolkit
302
available (Frank et al. 2005; Reski and Frank 2005; Quatrano
et al. 2007). It is also important from a phylogenetic perspec-
tive (Rensing et al. 2002; Cove et al. 2006), bridging the
evolutionary gap between sequenced green algae and
flowering plants, and thus making it a suitable organism for
plant evo-devo studies (e.g., Tanahashi et al. 2005; Menand
et al. 2007; Mosquna et al. 2009; Khandelwal et al. 2010).
In the light of what we have outlined above, P. patens and
its siblings also appears now as a model ideally suited to study
genome evolution through polyploidization and hybridization
in haploid-dominant organisms. Moreover, due to the exten-
sive range of morphological differences of the sporophyte,
ranging from the elaborate F. hygrometrica to the vastly
reduced P. patens morphology, Funariaceae are an excellent
model to study gene-phene evolution. The latter is made even
more interesting since the reduced sporphyte structure (as
observed in P. patens) has evolved independently several
times and might very well be related to a different mode of
propagation (predominantly selfing, spores resting in the
ground) in contrast to the cosmopolitan F. hygrometrica
(which produces numerous small spores suitable for long
range dispersal, the species propagating weed-like). The fact
that there is a low breeding barrier among the family also
enables crossing and associated genetic studies.
For Funariaceae to enter the age of population genomics
we will need to re-sequence transcriptomes and genomes of
other species and accessions from this family. Indeed, first
steps have already been made by genome and transcriptome
sequencing of P. patens isolates from geologically distinct
locations, such as the French Villersexel accession (avail-
able through the cosmoss.org genome browser), or are
progressing for cryptic species from Africa and Australia.
In addition, the transcriptome of F. hygrometrica has
recently been sequenced, mapped against P. patens as refer-
ence, and used for comparison of gametophyte and sporo-
phyte biased gene expression in comparison with
Arabidopsis thaliana (Szovenyi et al. 2010).
P. patens is but the first moss and bryophyte genome, but we
certainly need more (Beike and Rensing 2010) in order to
detect trends that cannot be hypothesized based on a single
species. Fortunately, the genome of Ceratodon purpureus,
which is already established as a genetic model (Cove and
Quatrano 2006), has been granted for sequencing by the US
Department of Energy. C. purpureus belongs to the Dicranidae
whereas P. patens is in the Funariidae. The last common
ancestor of the two lineages lived approximately 200 Mya
(Newton et al. 2007). This offers an evolutionary horizon
somewhere between the monocot-eudicot (~90–300 Mya)
and angiosperm-gymnosperm (~290–385 Mya) divergence
(Zimmer et al. 2007). In the same way that the comparative
analysis of the P. patens genome has helped to understand the
water-to-land-transition of plant life (Rensing et al. 2008), we
can expect the C. purpureus genome to greatly inform
S.A. Rensing et al.
comparative evolutionary studies of later steps in (moss) evo-
lution. The availability of a second moss genome from a
different subclass will also aid in discovering genomic traits
that are common to mosses or unique to either lineage. Meta-
bolic pathways in general seem to be more versatile and more
redundantly encoded in mosses than in seed plants (Oliver et al.
2004; Lang et al. 2005; Rensing et al. 2007, 2008). The genome
of a second moss will thus help in determining novel protein
functions in this regard, and aid in the detection of metabolic
pathways so far unknown among land plants. Also, C.
purpureus is a dioicous, obligate outbreeder and harbours sex
chromosomes (McDaniel 2005), so this will provide a further
interesting dimension to compare with the P. patens genome
that lacks them.
Finally, what about liverworts, the earliest branching
division among land plants (Qiu et al. 2006) and sister to
mosses: are they different from mosses in terms of genome
evolution? The most common chromosome counts among
liverworts (Marchantiophyta) are n ¼ 8 and n ¼ 9 and only
a very few species are reported to have higher chromosome
counts, like e.g., n ¼ 16, 18 or n ¼ 36 (Fritsch 1991). The
genus Marchantia is reported to have a quite uniform num-
ber of 8 or 9 chromosomes although there are exceptions i.e.,
Marchantia grisea (n ¼ 9 for male, n ¼ 10 for female),
M. breviloba (n ¼ 18), M. globosa (n ¼ 18), and M.
planiloba (n ¼ 8, n ¼ 16 þ 2). The genome of Marchantia
polymorpha is currently being sequenced by the US Depart-
ment of Energy and many chromosome counts are available
for isolates from Japan, Europe, and America. They mainly
confirm a chromosome number of eight or nine for this
widely distributed species, with the single exceptions of
n ¼ 10–18 (Fritsch 1991). Thus, genome plasticity as
measured by chromosome numbers seems to be significantly
lower in liverworts than in mosses. The forthcoming analysis
of the M. polymorpha genome will reveal whether it is a
paleopolyploid. If it is not, as might be expected based on the
narrow range of eight or nine chromosomes among
liverworts, the genome analyses might reveal some surprises
with regard to the evolution of this most ancient group of
haploid-dominant plants.
Acknowledgments We are grateful to Stanislav Karnatsevych for
conducting literature and experimental research with regard to crossing
and hybridization within the Funariaceae.
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 Genome Size Diversity and Evolution
in Land Plants 19
Ilia J. Leitch and Andrew R. Leitch

Contents 19.1 Introduction

19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
19.1.1 Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 The amount of DNA in the nucleus of a cell is commonly
19.2 Genome Size Data and Diversity Across Land Plants 308 referred to as the genome size or C-value and people have
19.2.1 Bryophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 been estimating this character in plants and animals for over
19.2.2 Lycophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 60 years. Today, with data available for over 7,000 species
19.2.3 Monilophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
19.2.4 Seed Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
(Table 19.1), land plants (embryophytes) are the best studied
19.2.5 Mechanisms Responsible for Generating Changes in of the major taxonomic groups of eukaryotes. This chapter
Genome Size in Different Land Plant Groups . . . . . . . . . . . 317 provides an overview of what is currently known about the
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 diversity of genome sizes encountered in land plant groups
and considers how such diversity might have evolved. The
chapter by Greilhuber and Leitch (2013, this volume) will
discuss the impact of this diversity on plants in terms of how
differences in genome size have an impact at all levels of
complexity, from the nucleus to the whole organism. This
chapter should also be read with reference to those by Weiss-
Schneeweiss and Schneeweiss (2013, this volume) who
explore intra- and inter-specific chromosome complexity
across angiosperms, including polyploidy and dysploidy,
Murray (2013, this volume) who discusses gymnosperm
chromosomes, and Barker (2013, this volume) who
considers the chromosomes of monilophytes and lycophytes.

19.1.1 Terminology

While the term ‘genome size’ has been broadly used in the
literature, it may refer to different things by different
people (Greilhuber et al. 2005). For example, it has been
used to refer to the amount of DNA in an unreplicated
monoploid chromosome set (n) or in a polyploid nucleus
where the DNA has been replicated. In an attempt to
stabilize the terminology and to bring greater precision
when referring to the amount of DNA in different phases
of the cell cycle (e.g., G1 and G2), different stages of the
I.J. Leitch (*) life cycle (e.g., haplophase and diplophase), and when
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey comparing the amount of DNA in a basic monoploid
TW9 3AB, UK
e-mail: i.leitch@kew.org chromosome set with the total amount of DNA in the

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 307

DOI 10.1007/978-3-7091-1160-4_19, # Springer-Verlag Wien 2013
308 I.J. Leitch and A.R. Leitch

Table 19.1 Genome size data available in land plants

No. of No. of species
species with genome Representation Min. Max. Mean Median Mode Range
recognised size data (%) (pg) (pg) (pg) (pg) (pg) (max./min.)
Non-vascular plants
Liverworts c. 5,000 43 0.9 0.21 7.97 1.2 0.8 0.76 38-fold
Mosses c. 12,000 184 1.5 0.17 2.05 0.5 0.4 0.45 12-fold
Hornworts c. 150 0 – – – – – – –
Vascular plants
Lycophytes c. 900 15 1.6 0.086 11.96 1.7 0.1 0.086 139-fold
Monilophytes c. 11,000 67 0.6 0.77 72.68 14.0 9.5 7.95 94-fold
Seed plants
Gymnosperms c. 850 204 24 2.25 36.0 18.6 7.9 10.0 16-fold

Angiosperms c. 352,000 6,287 1.8 0.065 152.23 5.9 2.5 0.60 2,342-fold

amount of DNA in an unreplicated, gametic nucleus with a

chromosome number n.

19.2 Genome Size Data and Diversity Across

Land Plants

Land plants first began to diverge around 450–460 million

Fig. 19.1 The different levels of DNA amount during the life cycle of
an angiosperm or gymnosperm
nucleus of a generative polyploid, Greilhuber et al. (2005)
proposed the following terminology which is adopted in
the current chapter.
The ‘Holoploid genome size’ or ‘C-value’ represents the
amount of DNA in the whole chromosome complement of
the nucleus with a chromosome number n, irrespective of the
degree of generative polyploidy.
The ‘Monoploid genome size’ refers to the amount of
DNA in one chromosome set of an organism. It is abbreviated
as the Cx-value. In non-polyploid organisms the C-value and
Cx-value are the same whereas in polyploids the Cx-value is
theoretical, derived by dividing the holoploid C-value by the
ploidy level.
In addition, for quantitative comparisons the terms should
always be used with a prefix number to indicate the amount
of DNA replication (Fig. 19.1). Thus a 1C-value refers to the
years ago (Mya) during the middle Ordovician (Kenrick and
Crane 1997; Rensing et al. 2008) and have since diversified
into four major groups: (1) the non-vascular bryophytes
which comprise liverworts, mosses and hornworts, (2) the
lycophytes, considered to be the first vascular plants
to evolve and are sister to the remaining vascular plants
(3) the monilophytes comprising marattioid and leptos-
porangiate ferns, horsetails (Equisetum), whisk ferns
(Psilotum, Tmesipteris) and ophioglossoid ferns (e.g.,
Ophioglossum, Botrychium) and (4) the seed plants compris-
ing angiosperms and gymnosperms. Knowledge of genome
sizes in these different groups is uneven and in the past this
has hampered studies aiming to compare genome size diver-
sity across land plants. Nevertheless, in recent years there has
been a huge increase in the amount of genome size data
available (particularly in the angiosperms, Bennett and
Leitch 2011) and this provides an opportunity to reassess
what it known about genome size diversity across land plants.
The following survey is based on genome size data available
in the Plant DNA C-values database (release 5.0, December
2010) which contains estimates for 7,058 land plant species
together with data not yet compiled into the database.
19 Genome Size Diversity and Evolution in Land Plants
Fig. 19.2 Chromosomes of (a) Genlisea aurea (1C ¼ 0.065 pg) and
(b) Paris japonica (1C ¼ 152.23 pg, 2n ¼ 40) taken at the same
magnification. (Image in (a) from Greilhuber et al. 2006 and (b) from
Pellicer et al. 2010)
The new data available have extended the range of
genome sizes encountered in land plants at both ends of
the scale so C-values now vary nearly 2,400-fold. Both
record holders are angiosperms with the smallest reported
in the eudicot genus Genlisea (G. aurea 1C ¼ 0.065 pg,
Fig. 19.2a) (Greilhuber et al. 2006; the report of 0.065 pg
for G. margaretae actually belongs to G. aurea;
J. Greilhuber, pers. comm.) while the largest to date is in
the monocot Paris japonica which is an octoploid with 2n
¼ 8x ¼ 40 and a staggering 1C ¼ 152.23 pg (Pellicer et al.
2010) (Fig. 19.2b).
Each group of land plants is characterized by a distinctive
genome size profile (Fig. 19.3) and to understand how these
may have evolved requires viewing the data within an evo-
lutionary framework. Fortunately, understanding the
relationships between and within the different groups
which comprise land plants has increased in recent years
and is outlined by Soltis and Soltis (2013, this volume).
Indeed, having such a framework is essential for providing
insights into the direction of genome size evolution in dif-
ferent groups.
19.2.1 Bryophytes
Traditionally, mosses (Bryophyta s.s.), liverworts (Marchan-
tiophyta) and hornworts (Anthocerotophyta) were grouped
together into the bryophytes, comprising the three extant
lineages of non-vascular land plants. However, they are no
longer considered to form a monophyletic group and instead
309
are recognised as three distinct paraphyletic lineages (Shaw
and Renzaglia 2004). Indeed, with increasing amounts of
both molecular sequence and structural data, several studies
suggest that liverworts are sister to all other land plants
followed by mosses, and with hornworts sister to all vascular
plants (tracheophytes) (e.g., Qiu et al. 2006; Pena et al. 2008;
Qiu 2008). Nevertheless, studies based on other sources of
data (e.g., sperm cell morphology) suggest alternative
relationships and more data are clearly needed to fully
resolve the branching order (Mishler and Kelch 2009).
Sadly there is not a great wealth of genome size data in the
bryophytes despite their key evolutionary position at the base
of all land plants. In addition, mosses are possibly the second
most diverse group of land plants after the angiosperms.
19.2.1.1 Liverworts
Until recently, genome size data were available for only nine
liverworts and even some of these values were called into
question (Voglmayr 2000; Temsch et al. 2010) due to
problems with methodology and material. For example, a
recent comparison between genome size estimates obtained
using Feulgen densitometry and flow cytometry highlighted
how only the later method may be considered reliable due to
the rigid cell walls and crystalline deposits in liverworts that
interfere with Feulgen staining (Temsch et al. 2010).
Fortunately, the publication by Temsch et al. (2010) of
genome size estimates for 43 species in 31 genera (32 foliose,
11 thallose) from 22 families has increased knowledge of
genome sizes in liverworts considerably. Although the data
are still far from representative given that there are c. 5,000
liverwort species worldwide comprising 83 families and 391
genera (Crandall-Stotler et al. 2009), some insights into
genome size diversity can now be gleaned. Overall, 1C-
values range 38-fold from 0.211 pg in Lejeunea cavifolia
(Lejeuneaceae) to 7.966 pg in Mylia taylorii (Myliaceae)
(Table 19.1) with no significant difference in the ranges
encountered in thallose (1C ¼ 0.293–7.966 pg) and foliose
(0.211–0.757 pg) liverworts (Temsch et al. 2010).
A histogram of the frequency of species with different
genome sizes shows that most liverworts have small
genomes (up to around 2 pg; Fig. 19.4a). Only two genera
have larger genomes – Pellia (Pelliaceae) and Mylia
(Myliaceae) and their genomes certainly appear as outliers.
What is notable about these species is that they belong to
different orders – Pellia to the Order Pelliales and Mylia
which is in Jungermanniales (Crandall-Stotler et al. 2009).
While there are currently no other genome size estimates for
species belonging to the other genus of Order Pelliales (i.e.,
Noteroclada), cytological studies show their chromosomes
are considerably smaller, suggesting much smaller genome
sizes in this genus (Proskauer 1950). In the Order
Jungermanniales several estimates are available and they
are all less than 1C ¼ 2.02 pg. Such observations suggest
310
Fig. 19.3 Histograms showing the genome size profiles for each of the major land plant groups with genome size data
that there have been two independent increases in genome
size within liverworts. The most extensive increase is in
Mylia taylorii as this species is reported to be haploid with
n ¼ 9. In Pellia, the largest genome is for a polyploid
(P. borealis with 1C ¼ 7.4 pg and n ¼ 18). Nevertheless,
its monoploid genome size (1Cx ¼ 3.7 pg) is similar in size
to related haploid species (with 1C ¼ 3.4–3.8 pg) which
themselves appear as outliers compared with other
liverworts (Fig. 19.4a).
Further large genomes may well be encountered in other
species of Mylia as M. anomala has been reported to have a
karyotype comprising eight similarly large chromosomes as
observed in M. taylorii, and these are said to be amongst
the largest known in liverworts (Newton 1987). As for Pellia,
large genomes may also be found in related species as large
chromosomes with lots of heterochromatin (up to 35%)
I.J. Leitch and A.R. Leitch
have been observed in P. neesiana (Newton 1985, 1987).
Large genomes may also be found in Pallavicinia lyellii
(Pallaviciniaceae) since cytogenetic studies by Zheng and
Zhu (2009) showed that this species had the same number
(n ¼ 8) but larger chromosomes than Pellia epiphylla. Sadly,
there are currently no genome size data for Pallavicinia.
19.2.1.2 Mosses
Mosses are the best represented group of bryophytes in terms
of genome size with values for 1.5% of species (Table 19.1).
Like liverworts, mosses are characterized by small genomes,
but they are considerably less variable, ranging just 12-fold
from 0.17 pg in Holomitrium arboretum to 2.05 pg in Mnium
marginatum (Fig. 19.4b). It is also noted that the distribution
of DNA amounts is strongly skewed towards the lower end of
19 Genome Size Diversity and Evolution in Land Plants
Fig. 19.4 (a, b) Histograms showing the distribution of 1C-values in
(a) 43 liverwort species and (b) 184 mosses. (c) The distribution of
C-values in the gametophytes of 39 species of the moss genus Sphag-
num showing the two distinct ploidy levels
the genome size range (Fig. 19.4b), so actually the majority of
moss genomes show an even narrower range, with 97% of the
data varying just five-fold from 1C ¼ 0.2–0.6 pg. It is possi-
ble that larger genomes may be uncovered, particularly in
species reported to have high chromosome numbers, which
may be polyploids (e.g., n ¼ 72 in Physcomitrium pyriforme
and Leptodictyum riparium – the highest chromosome num-
ber so far reported for a moss; Przywara and Kuta 1995).
However, since increasing levels of polyploidy may be
accompanied by decreases in chromosome size and genome
size, as noted in some liverworts (Bornefeld and Grillenberger
1987; Orzechowska et al. 2010) and some mosses (Abderrahman
2004), whether genomes larger than the largest liverworts will
be found in mosses is uncertain.
Among the mosses, data are available for 80 genera and 36
families although 96% of the genera and 75% of the families
have five or fewer genome size estimates. Most data are avail-
able for Sphagnum (the only genus comprising Sphagnaceae)
with estimates for 39 species. While C-values here range
0.39–0.95 pg, the data clearly fall into two groups which
have been shown to correspond to species whose gametophytes
are haploid (n ¼ 19) or diploid (n ¼ 38) (Temsch et al. 1998;
311
Greilhuber et al. 2003) (Fig. 19.4c). Such studies have
highlighted the apparent stability of the monoploid genome
size in Sphagnum (mean 1Cx ¼ 0.45 pg, S.D. 0.03) although
it remains to be determined whether this is typical for other
moss genera as insufficient data are currently available.
19.2.1.3 Hornworts
Currently, there are no reliable genome size data for hornworts.
Data for two species were reported by Renzaglia et al. (1995)
(i.e., Notothylas orbicularis 1C ¼ 0.17 pg, Phaeoceros laevis
1C ¼ 0.27 pg) and their values are both at the lower end of
those found in either mosses or liverworts. However, the Feul-
gen microdensitometry technique used to measure genome size
did not follow best practice recommendations (e.g., unsuitable
storage of nuclei prior to analysis, use of animal cells as
calibration standards, use of highly condensed sperm nuclei,
Temsch et al. 1998; Voglmayr 2000). In addition, the rigid cell
walls are likely to prevent access to reagents as noted for
liverworts above and hence to unreliable data (Greilhuber
pers. comm.).
Nevertheless, very small genomes might be expected
given that hornworts are characterized cytologically by
possessing low numbers of extremely small chromosomes
(n ¼ 4–10) and polyploidy is rare or absent (Proskauer
1958, 1967; Kuta and Przywara 2000). Clearly it is impera-
tive that genome size data are obtained for this important
group of non-vascular land plants, particularly if they are
indeed shown to hold a key phylogenetic position as sister to
all vascular plants as suggested by some molecular studies
(Qiu et al. 2006).
19.2.2 Lycophytes
Lycophytes which comprise c. 900 species are divided into
three lineages (clubmosses and firmosses – Lycopodiaceae,
quillworts – Isoetaceae and spikemosses – Selaginellaceae)
and have consistently been shown to be monophyletic and
sister to all other vascular plants using both molecular and
morphological characters (Schneider et al. 2009; Gao et al.
2010). They are therefore considered to hold a key position
in plant evolution. Sadly from a genome size perspective
they are poorly represented with data for just 15 species
although all three families are represented (Fig. 19.5). The
smallest genomes (1C ¼ 0.086–0.24 pg; 10 species) are
found in Selaginella (the only genus comprising
Selaginellaceae), which is also noted to be characterized by
possessing some of the smallest chromosomes in non-seed
vascular plants. In addition, most species are diploid with
2n ¼ 18 or 20 (Jermy 1967; Takamiya 1993). It therefore
seems likely that this family is characterized by a narrow
range of small genomes.
In contrast, a larger range of genome sizes is encountered
in Isoetaceae (comprising a single genus Isoetes) with values
312
Fig. 19.5 Distribution of
genome sizes in the three families
which comprise lycophytes. Data
available for 10 species in
Selaginellaceae, two species in
Isoetaceae and three species in
Lycopodiaceae
varying seven-fold (i.e., 1C ¼ 1.75 and 11.97 pg), and
including the largest genome so far reported for any
lycophyte (Isoetes lacustris, 1C ¼ 11.97 pg and 2n ¼ c.
110, Hanson and Leitch 2002). Even though data are cur-
rently available for just two species, this diversity reflects
what is known from cytological studies as some of the
species have been reported to have the largest chromosomes
in lycophytes (Dunlop 1949) while other studies have
revealed considerable variation in both chromosome number
and size between species (2n ¼ 22 – c. 130 and sizes of
individual chromosomes from less than 2 to 7–8 mm long,
Dunlop 1949; Manton 1950; Troı̀a 2001; Peruzzi et al.
2003).
Lycopodiaceae have genome sizes ranging from 1C
¼ 2.7–5.7 pg. However these are based on data for just
three species and only one chromosome count (i.e., Lycopo-
dium clavatum, 2n ¼ 68). Since this group of homosporous
lycopods can reach high chromosome numbers (up to 2n ¼ c.
510 in Phylloglossum (Blackwood 1953) and 2n ¼ c. 556 in
Huperzia prolifera (Tindale and Roy 2002) – the highest for
any lycophytes) larger genomes may be encountered as data
increase. However, genomes as large as some Isoetaceae
seem unlikely in all but Phylloglossum since it has been
suggested that there are constraints on genome size in groups
with biflagellate sperm (i.e., Lycopodiaceae (excluding
Phylloglossum), Selaginellaceae and all non-vascular plants,
Renzaglia et al. 1995). The hypothesis states that for species
with only two flagella there is an upper limit to genome size
determined by the efficiency with which the flagella can
I.J. Leitch and A.R. Leitch
move the sperm from the antheridia to the archegonia to
effect fertilization. Due to the positive correlation between
genome size and sperm size, an increase in DNA amount
would result in larger, less motile sperm, which may be
selected against. Multiflagellate sperm, which are more
mobile than biflagellate sperm, may not be so impeded and
thus genome size is not expected to be under such tight
selection pressure. Indeed, as noted above the largest
lycophyte genomes so far reported are indeed in Isoetaceae
which have c. 20 flagella per sperm (Renzaglia et al. 1995).
19.2.3 Monilophytes
Monilophytes are estimated to comprise c. 11,000 species
and data for 67 species show their genomes range 94-fold
from 1C ¼ 0.77 pg in the water fern Azolla microphylla to
72.68 pg in the whisk fern Psilotum nudum (Table 19.1). Yet
an analysis of the distribution of genome sizes (Fig. 19.6a)
shows that three of the five groups comprising monilophytes
(i.e., horsetails, marattioides and leptosporangiate ferns)
are characterized by small to medium sized genomes. The
very large genomes are restricted to species in the remaining
two groups of ferns – whisk ferns and ophioglossoid ferns.
Indeed their enormous genomes are clearly outliers, as they
are more than twice the size of the next largest monilophyte
genome which is in the horsetail Equistum variegatum with
1C¼ 30.3 pg (Fig. 19.6b).
19 Genome Size Diversity and Evolution in Land Plants
Fig. 19.6 (a) Distribution of genome sizes (1C-values) superimposed
onto a phylogenetic tree showing relationships between the five major
lineages of monilophytes. (b) Histogram showing the frequency of
genome sizes in 67 monilophyte species
While the monophyly of monilophytes is consistently
recovered in phylogenetic studies using molecular data and/
or morphological characters from both extant and fossil mate-
rial (Pryer et al. 2001; Qiu 2008; Schneider et al. 2009), the
relationships within the five clades comprising monilophytes
are still far from resolved (Schuettpelz and Pryer 2008;
Schneider et al. 2009). Yet whisk ferns and ophioglossoid
ferns are frequently grouped together and resolved as being
distinct from and sister to the remaining monilophytes.
Intriguingly, the very large genomes encountered in these
two groups appear to have evolved via two different cytologi-
cal mechanisms. Ophioglossoid ferns such as Ophioglossum
are characterized by possessing numerous small
chromosomes (1.5–4.5 mm in length, Abraham et al. 1962)
potentially arising through multiple rounds of polyploidy and/
or chromosome fragmentation. Indeed a chromosome count
of 2n ¼ c. 1,440 which may be 96-ploid has been reported in
O. reticulatum and is the highest chromosome number ever
reported for a plant (Khandelwal 1990). The genome size data
available for two species of Ophioglossum have counts of
2n ¼ c. 720 and c. 960 for O. gramineum and O. petiolatum
respectively. In contrast, the large genomes of the whisk ferns
313
(Psilotaceae – comprising Psilotum and Tmesipteris) have
evolved through increases in chromosome size as cytological
data show these genera are characterized by lower numbers
(i.e., 2n ¼ 104, 156, 208, 416) of larger (4.5–18 mm)
chromosomes with a maximum ploidy level of 8x (Manton
1950; Abraham et al. 1962; Brownsey and Lovis 1987).
At the other end of the scale the smallest monilophyte
genome so far reported is in the water fern Azolla
microphylla which belongs to a derived group of
leptosporangiate ferns. Its genome size was estimated to be
1C ¼ 0.77 pg with 2n ¼ 44 (the lowest number reported for
the genus, Stergianou and Fowler 1990; Hanson and Leitch
2002). Currently this is the only genome size estimate for
water ferns although Azolla is reported to have the smallest
chromosomes of any fern (<0.5 mm in A. pinnata also with
2n ¼ 44, Stergianou and Fowler 1990) so very small
genomes may well be found in related species. However,
bigger genomes are expected in the four other genera com-
prising water ferns (i.e., Salvinia, Pilularia, Marsilea and
Regnellidium) as cytological studies show they possess
larger chromosomes. In Salvinia which is considered sister
to Azolla and represents the other genus of Salvineaceae
(Nagalingum et al. 2008) chromosomes were 3–4 mm for
species with 2n ¼ 18 (Tatuno and Takei 1969), while in
the three genera comprising Marsileaceae chromosomes
varied with 4–8 mm in Regnellidium with 2n ¼ 38 (Loyal
1962), 3–5 mm in Pilularia with 2n ¼ 20 (Large and
Braggins 1989) and 1.5–3 mm in Marsilea with 2n ¼ 50
(Lesho 1994).
The best represented group of monilophytes is the
horsetails (Equisitum) where 10 out of the c. 15 recognized
species have genome size estimates. In addition, the phylo-
genetic relationships and species comprising the two
subgenera Hippochaete and Equisetum are now becoming
better resolved (Guillon 2004, 2007) enabling genome size
data to be considered within a phylogenetic context. All
species (except one reported triploid hybrid) have a chromo-
some count of 2n ¼ 216 but genome sizes range from
1C ¼ 12.5–30.3 pg (Fig. 19.7). Nevertheless, when the
data are superimposed onto the phylogenetic tree of Guillon
(2007) it is clear that they fall into two groups with the
species in subgenus Hippochaete characterized by larger
genomes (1C ¼ 20.7–30.3 pg) than those of subgenus Equi-
setum (1C ¼ 12.5–14.2 pg) (Fig. 19.7). These observations
fit with Manton’s (1950) cytological observations that spe-
cies belonging to subgenus Hippochaete were characterized
by larger chromosomes than those in subgenus Equisetum.
The recent resolution of the placement of E. bogotense
within subgenus Hippochaete (Guillon 2007) is also consis-
tent with the genome size estimate of this species of 1C
¼ 20.7 pg which is similar to E. scirpoides, another early
diverging species in the subgenus (Fig. 19.7).
314
Fig. 19.7 Distribution of genome sizes encountered in Equisetum superimposed onto the phylogenetic tree of Guillon (2007)
19.2.4 Seed Plants
Seed plants are estimated to comprise c. 96% of extant
vascular plant diversity and are divided into gymnosperms
(comprising c. 850 species) and angiosperms with an
estimated 352,000 species (Paton et al. 2008).
19.2.4.1 Gymnosperms
Gymnosperms first appeared in the fossil record in the Upper
Devonian, c. 350 Mya but while there are arguably 14 gym-
nosperm groups recognized, only four are represented in
extant floras (Bateman et al. 2006). These comprise the
cycads (c. 250 species), Ginkgo (1 species), Gnetales (c. 50
species) and conifers (c. 550 species) which are typically
divided into (1) Pinaceae and (2) the remaining non-Pinaceae
conifers. Relationships among the different gymnosperm
groups remain enigmatic, especially the position of Gnetales.
Molecular data point to affinities with the conifers (e.g., Chaw
et al. 2000; Werner et al. 2009), while the analysis of mor-
phological characters provides little support for such a rela-
tionship (Palmer et al. 2004) (see also Sect. 14.1 in Murray
2013, this volume and Soltis and Soltis 2013, this volume).
From a genome size perspective, gymnosperms represent
the group of land plants with by far the best representation of
I.J. Leitch and A.R. Leitch
data. There are C-values for c. 24% of all described species
(Table 19.1) including at least one genome size estimate for
each of the 13 families recognized. While gymnosperm
genomes are, on average, larger than other land plant groups,
especially the angiosperms (mean 1C ¼ 18.6 pg compared
with angiosperms where mean 1C ¼ 5.9 pg: Table 19.1),
gymnosperms remain one of the least variable groups with
DNA amounts ranging just 16-fold from 2.25 pg in Gnetum
ula to 36.0 pg in Pinus ayacahuite. In addition, the genome
size profile is not strongly skewed towards small DNA
amounts, unlike liverworts, mosses, lycophytes and
angiosperms (Fig. 19.3).
Cycads, which are often considered to be sister to all
other extant gymnosperms, have medium-sized genomes
(1C ¼ 12.01–21.1 pg; Fig. 19.8a). While this is based on
data for just six species it is consistent with the more exten-
sive chromosomal data showing this group is typically
characterized by large chromosomes with a narrow range
of numbers (2n ¼ 16–28, e.g., Sax and Beal 1934; Marchant
1968, see also Murray 2013, this volume).
Gnetales comprise three phylogenetically distinct
families – Gnetaceae, Ephedraceae and Welwitschiaceae,
all of which are monogeneric. They have genomes ranging
8-fold from 1C ¼ 2.25–18.22 pg (Fig. 19.8a). The smallest
19 Genome Size Diversity and Evolution in Land Plants
Fig. 19.8 (a) Distribution of genome sizes within the four groups of
gymnosperms. Numbers in brackets correspond to the number of spe-
cies in each group with genome size data. Arrows point to the mean 1C-
value for each group (where applicable). (b) Comparison of the mean
(•) and range of genome sizes in each of the 13 families comprising
gymnosperms. Families are arranged into their higher order groups but
within each group there is no phylogenetic order. The number in
brackets following the family name corresponds to the number of
species with genome size data
genomes of any gymnosperm are found in Gnetum
(Gnetaceae) with 1C ¼ 2.25–3.98 pg and chromosome
numbers of 2n ¼ 22 or 44, representing diploid and tetra-
ploid species. Welwitschiaceae comprises a single species,
Welwitschia mirabilis, and has an intermediate-sized
genome of 1C ¼ 7.2 pg and 2n ¼ 42. Most variable in
Gnetales is Ephedra (Ephedraceae) with genome sizes rang-
ing from 1C ¼ 8.8–18.22 pg. This reflects the occurrence of
both diploid (2n ¼ 14) and tetraploid (2n ¼ 28) species
with genome size data (Fig. 19.8a). Indeed, although poly-
ploidy is very rare in gymnosperms as a whole, Ephedra is
distinctive as 44% of species are reported to be polyploid
(Delevoryas 1980). (N.B. one hexaploid (E. distachya
subsp. distachya) and two octoploids (E. gerardiana and
E. funerea) have been reported by Ickert-Bond (2003) but
315
no genome size data are currently available for these
cytotypes.)
The greatest diversity of genome sizes are found in the
conifers with values ranging from 1C ¼ 6.6 to 36.0 pg
(Fig. 19.8a and b). However, most of this variation is
found in Pinus (Pinaceae, 1C ¼ 16.6–36.0 pg) which is the
most variable genus of any gymnosperm despite all species
having the same chromosome number of 2n ¼ 24 (see
Murray 2013, this volume). The only other notably large
genome is that of Sequoia sempervirens with 1C ¼ 32.1 pg.
Its genome is clearly an outlier compared with the
genome sizes encountered in other species of the non-
Pinaceae group of conifers (Fig. 19.8a). The large
genome of S. sempervirens is in part due to polyploidy as
cytologically it is a hexaploid with 2n ¼ 6x ¼ 66, with the
highest chromosome number reported to date for any
gymnosperm.
19.2.4.2 Angiosperms
Given the astonishing ecological, species and genomic
diversity of angiosperms, insights into their genome sizes
has long attracted the attention of scientists. Indeed, the first
plant to have its genome size measured was an angiosperm
Lilium longiflorum in 1951 (Ogur et al. 1951). Since then
many species have had their genome sizes estimated and
there are currently values for 6,287 species in the Plant DNA
C-values database (Bennett and Leitch 2010). As already
mentioned in Sect. 19.2, the most striking feature of
angiosperms is the huge diversity of genome sizes encoun-
tered (Figs. 19.2 and 19.3, Table 19.1), considerably greater
than any other group of land plants and extending nearly
2,400-fold. In addition, the genome size profile is quite
distinct from that of other land plant groups (Fig. 19.3),
with most species characterized by having small to very
small genomes. Indeed, the modal and median 1C-values
for 6,287 species are just 0.6 and 2.5 pg respectively. Species
with large to very large genomes are comparatively few and
localized in just a few lineages.
Over the years, insights into how this genome size diver-
sity is spread across the phylogenetic tree of angiosperms
have been gleaned by superimposing existing data onto
phylogenetic trees available at the time of analysis (e.g.,
Leitch et al. 1998; Soltis et al. 2003; Leitch et al. 2005).
The most recent of these was based on data for 4,119 species
using the phylogenetic tree published by the Angiosperm
Phylogeny Group (APG) in 2003 (APG II 2003). Given a
52% increase in the amount of genome size data available
since then, together with an increasingly well resolved phy-
logenetic tree of the angiosperms (Soltis et al. 2008; APG III
2009), the data have been updated and reanalysed here
(Fig. 19.9).
The overall picture, which supports previous studies,
shows that all angiosperm groups contain species with
small to very small genomes, the extremely large genomes
are phylogenetically restricted to some species belonging to
316
Fig. 19.9 Distribution of genome size across angiosperms
superimposed on the phylogenetic tree given in APG III (2009).
C-value data for each group are shown as a line connecting the
minimum and maximum 1C-value with the mean shown as •. The
just two groups – the Santalales (within eudicots) and
monocots (Fig. 19.9). A closer look shows that even within
these groups, species with very large genomes occupy
derived positions within the phylogenetic tree (Figs. 19.10
and 19.11).
In Santalales a histogram (Fig. 19.10a) shows that most
are characterised by small to medium genomes, the four very
large 1C-values (1C ¼ 62.3–82.0 pg) all belong to Viscum
species and are clearly outliers. Nevertheless, as data repre-
sentation improves, further similarly large genomes may
indeed be found in other genera of Viscaceae as cytological
studies have shown that related genera Notothixos,
Dendrophthora and Phoradendron contain species with sim-
ilar numbers of extremely large chromosomes (e.g.,
Fig. 19.10b, Wiens 1964; Wiens and Barlow 1971). So far
the species of Viscum which have had their genome sizes
estimated have either been diploid with 2n ¼ 20 (V. album),
24 (V. crassulae) or 28 (V. minimum) or assumed to be based
on a similar genome size to the related diploids (i.e., V.
cruciatum). Although polyploidy is rare in this genus and
in the familiy Viscaceae as a whole, a few polyploids of
Viscum and Phoradendron have been reported (Wiens and
Barlow 1971) suggesting that considerably larger genomes
could be found in this clade of eudicots.
I.J. Leitch and A.R. Leitch
number in brackets following the group name gives the number of
species with C-value data. Major subdivisions of the angiosperms are
shown on the right (Basal angios. basal angiosperms)
Recent improvements in the understanding of phylogenetic
relationships within Santalales by Nickrent et al. (2010) shows
that Viscaceae is deeply embedded within the Santalales tree
suggesting that the large genomes are derived rather than
plesiomorphic for the order. Indeed other genera within
Viscaceae are characterised by possessing smaller chromo-
somes (e.g., Arceuthobium), although these are still consider-
ably larger than is typical of many angiosperms (Wiens 1968).
An independent increase in genome size may also have occurred
in Loranthaceae, another family in Santalales. Although the
available genome size for 57 species in this family are all
small to medium sized (1C ¼ 1.6–17.5 pg), once again chro-
mosome data suggest the presence of much larger genomes
(Wiens 1964). Thus while the early diverging genera of
Loranthaceae, Nuytsia, Atkinsonia and Gaiadendron, have rela-
tively small chromosomes and genomes (i.e., Nuytsia 1C ¼ 2.9
pg) (Barlow and Wiens 1971; Martin 1983), very large
chromosomes have been reported in Aetanthus, Struthanthus
and Psittacanthus (Fig. 19.10b, Wiens 1964) all of which belong
to tribe Psittacanthinae (sensu Nickrent et al. 2010). Indeed, as
early as 1946 Covas and Schnack (1946) suggested that
Psittacanthus possessed the largest chromosomes in the
eudicots. As the chromosome numbers of these genera are
2n ¼ 16 it remains to be seen whether their genomes are larger
19 Genome Size Diversity and Evolution in Land Plants
Fig. 19.10 (a) Histogram showing the distribution of genome sizes for
64 species of Santalales. (b) Meiotic chromosomes of four species in
Santalales reported to have large chromosomes, all reproduced at the
same magnification (Images for Phoradendron, Struthanthus and
Psittacanthus taken from Wiens 1964 and reproduced with permission)
than those in Viscum or indeed Paris japonica, the latter with the
largest known eukaryote genome (Pellicer et al. 2010).
This pattern is repeated within monocots (Fig. 19.11). Here
genome size profiles of all the major groups show most
monocots are characterized by small genomes (Leitch et al.
2010). Species with very large genomes are restricted to just a
few genera within Asparagales and Liliales, which are well
embedded within the monocot tree (Fig. 19.11). The most
unusual distribution of genome sizes is found within Liliales
where an analysis of 145 species from seven families showed a
100-fold range in genome sizes (1C ¼ 1.53–152.23 pg), and
no clear skew in the data towards species with small genomes
as observed in most other angiosperm groups (Figs. 19.9 and
317
19.11). The truly enormous genomes greater than 1C ¼ 100 pg
are only found in three genera belonging to Liliales: Paris and
Trillium (Melanthiaceae) and Fritillaria (Liliaceae) (Pellicer
et al. 2010; Zonneveld 2010).
19.2.5 Mechanisms Responsible for Generating
Changes in Genome Size in Different
Land Plant Groups
19.2.5.1 Angiosperms
Most of our understanding of the mechanisms responsible
for changes in genome size has come from studies of
angiosperms (for recent reviews see Kejnovsky et al. 2009;
Grover and Wendel 2010). It is recognised that increases in
genome size arise predominantly through polyploidy and
amplification of non-coding repetitive DNA, especially
retrotransposons. These mechanisms are counterbalanced
by processes that result in a decrease in genome size.
These are less well understood, but have been shown to
involve recombination-based processes for example unequal
recombination and illegitimate recombination (reviewed by
Grover and Wendel 2010).
While polyploidy is widely considered to play a role in
generating increases in genome size, its long-term impact on
genome size evolution is less clear and may be subject to
different selection pressures in the different land plant
groups. In angiosperms, while genome size increases imme-
diately following a polyploid event, an extensive survey of
genome size data for 3,021 species showed that this is often
accompanied over time by ‘genome downsizing’ (Leitch and
Bennett 2004). This is supported by more focused compara-
tive genomic studies which have shown how extensive loss
of DNA can take place in specific species. For example, in
maize and rice it is estimated that over 50% of the genes
have been lost since the last round of polyploidy in their
ancestries (e.g., Messing et al. 2004; Wang et al. 2005).
These observations suggest that multiple rounds of poly-
ploidy do not inevitably lead to larger genomes (Leitch and
Bennett 2004), indeed many angiosperms with very big
genomes are not cytological polyploids (e.g., Fritillaria
koidzumiana 2n ¼ 2x ¼ 24, 1C ¼ 77.5 pg).
19.2.5.2 Gymnosperms
The mechanisms involved in generating the large genomes
typical of many but not all gymnosperms have not been
investigated as extensively as angiosperms but preliminary
data suggest there are differences. Overall polyploidy, as
identified through chromosome numbers is rare in
gymnosperms, with only 5% of species estimated to be
polyploid, nearly all of which are in Ephedra (Delevoryas
1980). Clearly, ongoing polyploidy in extant gymnosperms
is not contributing to the large genomes typical of this group
of seed plants. Nevertheless, emerging from the increasing
318 I.J. Leitch and A.R. Leitch

Fig. 19.11 The distribution of a b

genome sizes for 2,530 species of 125 Poales
100
monocots. (a) The summary 75 (0.2 - 26.0 pg)
50
25
0
topology of monocots based on 40
30 Zingiberales
Chase et al. (2006) with the Poales (951) 20
10 (0.3 - 6.0 pg)
number in brackets corresponding 0

to the number of species with Dasypogonaceae (1) 10

8
Commelinales
6
4 (0.8 - 43.4 pg)
genome size data. (b) Histograms Zingiberales (71) 2
0
showing the distribution of Commelinales (113)
25
20
Arecales
15
(0.9 - 30.0 pg)
genome sizes within each order Arecales (89)
10
5
0
with the range of 1C-values in 125
100
Asparagales
brackets. (Figure modified from Asparagales (1130)
75
50
25
(0.3 - 82.2 pg)
Leitch et al. 2010 and reproduced 0
10 Liliales
8
with permission) 6
4 (1.5 - 152.2 pg)
Liliales (145) 2
0
10
Pandanales (8) 8 Pandanales
6
4
2
(0.4 - 1.5 pg)
Dioscoreales (14) 0
10
8 Dioscoreales
Petrosaviales (0) 6
4
2
(0.4 - 6.8 pg)
Alismatales (106) 0
20
15 Alismatales
10
Acorales (2) 5 (0.3 - 24.1 pg)
0
0 25 50 75 100 125 150
1C DNA amount (pg)

amounts of genomic and transcriptomic sequence data is
tentative evidence that at least some gymnosperm groups
have undergone whole genome duplications in their evolu-
tionary history (Cui et al. 2006; Barker et al. 2010).
For conifers, retrotransposon amplification is clearly an
important contributor to genome expansion. This is based on
the increasing amount of data showing that most of their
DNA has originated through the amplification of a large
diversity of retrotransposons, present in a huge range of
copy numbers (Elsik and Williams 2000; Morse et al.
2009). In addition, recent data suggest that many of these
retrotransposons are still actively transcribing (Morse et al.
2009; Parchman et al. 2010). Perhaps conifers are less effi-
cient at suppressing retrotransposon amplification than
angiosperms (see review by Leitch and Leitch 2012).
Mechanisms of DNA elimination are poorly understood
in gymnosperms. A recent study of one retroelement in
Pinus (Ty-gypsy element Gymny) suggested there was little
recombination between the long terminal repeats (LTRs) of
this element (Morse et al. 2009). As this type of recombina-
tion has been shown to contribute to DNA loss in
angiosperms (Vitte and Panaud 2003; Vitte and Bennetzen
2006), the data may indicate that this mode of genome
downsizing is not contributing extensively to eliminating
amplified retrotransposons in gymnosperms and may further
explain why their genomes are, on average larger than those
of angiosperms.
Sadly there are currently little data available for non-
conifer gymnosperms especially Gnetum which has the
smallest genomes (Fig. 19.8) and Ephedra where polyploidy
is more common. Indeed, investigations into the genomic
composition of these non-conifer gymnosperms are essential
to provide broader insights into the genomic diversity of
gymnosperms and whether the picture emerging from the
study of conifers is representative for gymnosperms as a
whole. Such data could also shed light on the currently
unresolved relationship between Gnetales and the rest of
the gymnosperms and angiosperms.
19.2.5.3 Monilophytes
The limited but increasing genomic data becoming available
for monilophytes suggest that the genomic organization and
evolution of their genomes are different from those of seed
plants. Monilophytes, particularly the homosporous ferns,
are typically characterized by possessing numerous small
chromosomes which are strongly bivalent pairing and
undergo relatively little recombination (Nakazato et al.,
2008) (see also Barker 2013, this volume)
Given the reported high chromosome numbers in
monilophytes, it has been suggested that polyploidy is the
predominant mechanism involved in genome size expansion
in this group. However, recent studies suggest that poly-
ploidy may not be as prevalent as once thought while in
depth genomic studies of the model fern Ceratopteris
richardii have shown that the evolution of the monilophyte
polyploid nucleus may follow a very different trajectory
from that in angiosperms.
There is now increasing evidence that the largest clade of
homosporous ferns (the polypod ferns) underwent a single
whole genome duplication event c. 180 Mya ago (Barker and
Wolf 2010). Yet analysis of the genomic structure of C.
richardii suggests this was not accompanied by extensive
chromosome loss or elimination of DNA, as reported in
angiosperms. Indeed, linkage map analysis of C. richardii
shows that its genome has one of the highest proportions of
multiple copy genes among plants (Nakazato et al. 2006).
19 Genome Size Diversity and Evolution in Land Plants
Instead, gene silencing appears to have been the more impor-
tant diploidization processes operating (Pichersky et al.
1990; Barker and Wolf 2010). These and related studies
have led to the suggestion that ferns have a lower rate of
whole genome duplication and experience chromosomal and
DNA loss at a much slower rate than angiosperms (Barker
and Wolf 2010). Together these factors may contribute to
explaining the less skewed distribution of genome sizes
observed in ferns compared with angiosperms (Fig. 19.3).
Very little is known about the contribution of repetitive
DNA especially retrotransposons to genome size diversity in
ferns and this seems an area ripe for further study. Limited
data have shown that ferns contain the major classes of
retrotransposons (Flavell et al. 1992; Voytas et al. 1992)
but their contribution to genome size remains unquantified.
Brandes et al. (1997) noted that the abundance of Ty-copia
elements in the leptosporangiate fern Pteris cretica was
lower than in other land plant groups studied, whereas in
the whisk fern Psilotum triquetrum not only were high copy
numbers estimated to be present but the sequences were also
shown to be extremely heterogeneous compared with the 56
other species studies (which included representatives from
all the land plant groups, Flavell et al. 1992). Clearly further
studies are needed to clarify the nature and contribution of
these elements to genome size diversity encountered in
monilophytes.
19.2.5.4 Bryophytes
Given the limited genomic data available for hornworts and
liverworts the mechanisms responsible for generating the
narrow genome sizes observed remain enigmatic. Neverthe-
less, it is clear that ongoing polyploidy is not a major evolu-
tionary process in these lineages given its rarity in liverworts
and particularly hornworts. As to whether paleopolyploidy
events played a role in shaping their genomes, this is cur-
rently unknown given the lack of genomic sequence data
available for these lineages. Nevetheless, insights may soon
be gleaned at least for liverworts given that the genome of
Marchantia polymorpha is currently being sequenced.
In contrast, polyploidy is reported to be common in
mosses and the release of the first complete genome
sequence of a moss Physcomitrella patens (1C ¼ 500 Mb,
Rensing et al. 2008) provides at least some molecular
insights into its genomic make up and possible factors
contributing to the narrow range of small genome sizes
encountered in mosses. These studies have shown that the
P. patens genome is not only a neopolyploid (possibly a
triploid with n ¼ 27) but also has undergone at least one
whole genome duplication c. 30–60 Mya (see also Rensing
et al. 2013, this volume). Clearly then polyploidy is an
important factor contributing to the genomic make up and
genome size of this species. Amplification of retrotrans-
posons has also clearly been occurring given that 48% of
319
its genome is made up of retrotransposons, one of the highest
percentages noted for any completely sequenced plant
genome to date (Lang et al. 2008). Indeed, evidence of
three peaks in retrotransposon activity over the last 3 million
years is apparent.
Given the prevalence of polyploidy and retrotransposons,
the narrow range of genome sizes in mosses is puzzling and
suggests that mechanisms leading to DNA elimination are
operating efficiently in moss genomes. In support of this is
the nearly complete absence of helitrons in the genome of
P. patens. These rolling-circle transposons are an ancient
class of transposable elements and have been found in all
eukaryotic genomes sequenced to date. The presence of just
one family of highly conserved helitrons in P. patens
suggests that this helitron family has been active in the last
3 million years and that the lack of other helitrons is due to
their rapid and efficient removal from the genome (Rensing
et al. 2008). In addition, the low abundance of complete full
length retrotransposons suggests that DNA elimination via
recombination is taking place, while the presence of an
extensive collection of siRNAs which map to
retrotransposons suggests that for most of the time
retrotransposons are kept in check by post-transcriptional
silencing. Analysis also suggests that local gene duplications
do not contribute significantly to the P. patens genome as
only 1% of protein coding genes were shown to occur in
tandem array. This figure is much lower than in angiosperm
genomes which have been completely sequenced such as
14% in Oryza sativa (International Rice Genome Sequenc-
ing Project 2005) and 11% in Populus trichocarpa (Tuskan
et al. 2006).
Conclusions
Outside of the angiosperms, and to a lesser extent
Pinaceae in the gymnosperms, we clearly know very little
of the mechanisms shaping genome structure and organi-
zation across land plants. Even within these groups we
have patchy knowledge. For example, amongst
angiosperms we have most in depth understanding of
just a few key model or crop species (especially Poaceae
and Brassicaceae). However, the limited available data
suggest that the mechanisms that shape genomes in land
plants (i.e., polyploidy, retroelement mobility and ampli-
fication, tandem repeat expansion and contraction,
unequal homologous and illegitimate recombination) are
likely to occur in all groups, but their relative activities
and associated selection pressures may vary significantly.
The most notable feature about genome size diversity
in land plants is the frequency of species with small
genomes. It is the exception rather than the rule to find
truly enormous genomes and these are phylogenetically
restricted to just a few angiosperms and ophioglossoid
monilophytes.
320
The small size of most plant genomes suggests an
overall strong selection pressure to limit their size. As to
the nature of the selection pressures, these may well be
different for each land plant group. For example, it has
been suggested that there may have been selection for
small genomes in the early diverging angiosperms as it
enabled them to compete more efficiently with the larger-
genomed gymnosperms because of their more rapid
development: small genomes correlate with several
developmental phenotypic characters such as short mini-
mum generation times and rapid establishment of
seedlings (Leitch et al. 2005; see also Greilhuber and
Leitch 2013, this volume). In contrast, as noted above
(Sect. 19.2.2), selection for a small genome in biflagellate
bryophytes and lycophytes may explain the low DNA
amounts encountered in these species.
One factor which could be relevant to all land plant
groups is the limitation in genome size imposed by the
metabolic costs of assimilating and building nucleic
acids. Such a constraint on genome size is likely to be
particularly acute for primary producers growing in soils
limited in the building blocks of DNA (i.e., phosphorous
and nitrogen, Hanson et al. 2001; Leitch and Leitch
2008). It is perhaps relevant that gymnosperms which
have a larger modal and mean genome size than other
land plant groups (Table 19.1) are found in parts of the
world that are limited by water rather than nutrients.
Typically gymnosperms are specialist of high montane,
arctic and sandy soils which are often considered to be
relatively nutrient-rich. It is also perhaps notable that the
smallest gymnosperm genomes belong to species of
Gnetum, the only genus of gymnosperm to grow in the
relatively nutrient-poor soils of the tropics.
As for the giant genomes of angiosperms and
ophioglossoid ferns, these need not necessarily be a
consequence of polyploidy, instead a major contributing
factor is likely to be the activity of retrotransposons
and other non-coding repetitive DNA sequences. It is
unknown whether the reason for the massive abundance
of repeats is due to a greater activity of the elements or
reduced activity of the genome surveillance mecha-
nisms which suppress their activity. Alternatively, it
may simply be different selection pressures acting on
similar levels of raw variation. With the rapid advances
being made in the speed at which sequence data is
generated through second and third generation sequenc-
ing technologies it is hoped that a phylogenetically
diverse range of species will be studied to provide far
reaching insights into the genomic basis behind the
immense diversity of genome sizes encountered in
land plants.
I.J. Leitch and A.R. Leitch
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 Genome Size and the Phenotype
20
Johann Greilhuber and Ilia J. Leitch

Contents 20.1 Introduction

20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
20.2 Basic Facts: The Correlation of Genome Size with
Land plant species (Embryophyta) vary more than 2,300-
Cell Size and Cell Cycle Time . . . . . . . . . . . . . . . . . . . . . . . . . . 324 fold in the size of the holoploid genome (C-value) (see
20.2.1 The Nucleo-Cytoplasmic Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . 324 Leitch and Leitch 2013, this volume) with the extremes at
20.2.2 Large Genomes Are Correlated with Long Nuclear both ends of the scale contributed by angiosperms. At the
Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
lower end we find some species of the carnivorous
20.3 Hypotheses About the Biological Significance Lentibulariaceae with ultrasmall genomes, e.g., Genlisea
of Genome Size Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
aurea with 0.065 pg or 63.5 Mb (1C) (Greilhuber et al.
20.3.1 Accessory DNA is Mostly of Selfish Origin and
Represents Inert Genetic Junk . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 2006). This is only about 0.4-fold the size of the genome
20.3.2 The Nucleotype Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 of Arabidopsis thaliana with 0.16 pg or 156.5 Mb (1C)
20.3.3 Nucleoskeletal DNA Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . 328 (Bennett et al. 2003), a species long considered to be the
20.3.4 Alternatives to the Nucleotype and Nucleoskeletal
plant with the smallest reliably determined genome size. At
Hypotheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
the upper end of the scale stands the octoploid monocot
20.4 Examples and Explanations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 Paris japonica (Melanthiaceae) with 2n ¼ 8x ¼ 40 and
20.4.1 Cell Size, Genome Size and Sperm Mobility
in Bryophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 152.23 pg or 148.88 Gb (1C) (Pellicer et al. 2010). Never-
20.4.2 The Impact of Genome Size on Phenology . . . . . . . . . . . . . 331 theless, other monocot species such as Fritillaria davisii
20.4.3 The Relationship Between Genome Size and Weediness 332 (69.45 pg, 1C; 2n ¼ 2x ¼ 24, a possible paleotetraploid),
20.4.4 Genome Size and Invasiveness . . . . . . . . . . . . . . . . . . . . . . . . . . 332 Trillium apetalon (95.0 pg, 1C; 2n ¼ 4x ¼ 20), and the
20.4.5 Genome Size and Climate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
20.4.6 Genome Size and Pollution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 dicot tree parasite Viscum album (102.9 pg, 1C; 2n ¼ 20,
20.4.7 The ‘Large Genome Constraint Hypothesis’ . . . . . . . . . . . . 335 paleotetraploid?) also rank high on the scale regarding
20.5 Intrapopulational and Intraspecific Variation
monoploid genome size (i.e., Cx-value ¼ 2C-value divided
of Genome Size: Adaptively Important? . . . . . . . . . . . . . . 336 by ploidy level) (Zonneveld 2010). From these examples it is
20.5.1 Evolution Canyon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 clear that polyploidy plays only a relatively small role in the
20.5.2 Festuca pallens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 origin of the huge genome size differences reported. Instead,
20.6 Conclusions and Future Outlook . . . . . . . . . . . . . . . . . . . . . . 340 it is the accumulation of retrotransposon-like and other
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
repetitive elements in the genomes which are largely respon-
sible for the huge diversity of genome sizes in plants
(Bennetzen et al. 2005; Grover and Wendel 2010; see also
Kejnovsky et al. 2012 in volume 1). (N.B. Recent studies
using flow cytometry to estimate genome size in species
with enormous genomes have highlighted how the more
traditional approach of estimation using Feulgen densitome-
try may considerably underestimate genome size at this
upper end of the scale (Zonneveld 2010).)
J. Greilhuber (*)
Interest in the diversity of genome sizes has a long
Department of Systematic and Evolutionary Botany, Faculty of Life history, dating back to the early cytological studies
Sciences, University of Vienna, A 1030 Vienna, Austria showing enormous variation in the size of cell nuclei and
e-mail: johann.greilhuber@univie.ac.at

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 323

DOI 10.1007/978-3-7091-1160-4_20, # Springer-Verlag Wien 2013
324
chromosomes between different organisms (Gulliver 1875;
Stebbins 1971). However, it was following the discovery of
DNA and its presence in the nucleus and chromosomes that
studies started to focus on estimating the amount of DNA in
cells of different tissues of the same organism and between
species (e.g., Boivin et al. 1948; Vendrely and Vendrely
1948; Swift 1950). Such research soon uncovered two nota-
ble observations; (1) there was a remarkable constancy in
genome size within a species, and (2) there was a surprising
variation in genome size between organisms. Indeed, just 15
years after the first genome size for a plant was estimated by
Ogur et al. (1951) it soon became clear that there was
considerable diversity in genome size even between species
and genera. For example, Rothfels et al. (1966) reported a
40-fold range of genome sizes in 22 diploid genera belong-
ing to Ranunculaceae, while Martin and Shanks (1966)
noted a five-fold variation within the genus Vicia.
The problem during this period (i.e., 1950s – late 1970s)
was that it was broadly assumed that the amount of DNA in
an organism reflected the number of genes it had, and yet
there appeared to be no clear correspondence between
genome size and organism complexity (taken as a proxy
for gene number). In 1971 Thomas coined the term ‘C-
value paradox’ to reflect this apparent lack of relationship
(Thomas 1971).
The solution to the ‘C-value paradox’ was largely found
with the advent of the molecular age, as it soon became clear
that most of the DNA in the genome is non-coding. Never-
theless, while the ‘paradox’ may be considered to have been
solved (i.e., genomes vary in size mainly due to differences
in the amount of non-coding DNA) there are still many
unanswered biological questions remaining (e.g., what
types of non-coding DNA sequences predominate in
genomes of different sizes? what are the mechanisms
involved in generating genome size variation? what are the
evolutionary forces driving genome size changes?), leading
Gregory (2001) to coin the phrase ‘C-value enigma’ to
reflect this.
Indeed, the biological causes behind genome size
differences and the consequences for the organism of having
a large or small genome size are topics of a research disci-
pline in its own right (Bennett 1971, 1972; Cavalier-Smith
1985; Bennett 1998; Gregory 2005; Leitch and Bennett
2007). This chapter outlines how genome size is correlated
with cell size and cell cycle time (Sect. 20.2), the various
hypotheses that have been put forward to explain these
correlations (Sect. 20.3) and examples of the impact of
genome size variation at the whole plant level (Sect. 20.4).
A necessary premise for the following discussions is to
mention the relative constancy of genome size within a
species. While many studies have shown that genome size
is relatively constant within a species, there have been
reports in the literature of extensive intraspecific variation.
J. Greilhuber and I.J. Leitch
Many of these studies have subsequently been shown to be
artefacts arising from poor technique (discussed by
Greilhuber 1998, 2005, 2008), but recent high resolution
flow cytometric studies demonstrate that intraspecific and
even intrapopulational variation (e.g., Šmarda and Bureš
2006; Šmarda et al. 2007; Slovák et al. 2009; Šmarda et al.
2010) does exist (mostly within 10%, and exceptionally up
to 1.37-fold) (reviewed in Šmarda and Bureš 2010). The
biological importance of such variation and it potential
adaptive significance is discussed in Sect. 20.5.
Another premise is that there will be a certain minimum
amount of DNA in a plant genome that is determined by the
number of necessary genes and regulatory sequences,
together with indispensible sequences which play an archi-
tectural role. This amount has been estimated to be about
50 Mb (Bennett and Leitch 2005). Plants such as Genlisea
aurea are within this order of magnitude. DNA above the
required minimum—here we call it accessory DNA—may
be inert, may have a function, or may merely have an effect
(in a similar way as waste may have an effect in human
society). Inert DNA is dispensable without disadvantage,
DNA with effect may appear dispensable but selectively
sensitive, while DNA with function is indispensible or at
least selectively strongly sensitive. To separate function
from effect is difficult, and unfortunately the possibility of
experimenting is very limited. We are therefore currently in
the situation where most evidence comes from correlations
of genome size with organismal characteristics and environ-
mental parameters, which leaves room for interpretations
about the causes involved.
20.2 Basic Facts: The Correlation of Genome
Size with Cell Size and Cell Cycle Time
20.2.1 The Nucleo-Cytoplasmic Ratio
Cytologists have been aware of the correlation between
nuclear size and cell size in animals and plants for over
130 years, with the first studies by Gulliver in 1875. These
observations led to the concept of the ‘nucleo-cytoplasmic
ratio’ (e.g., Strasburger 1893) or the ‘karyoplasmic ratio’
(e.g., Hertwig 1903) where the ratio of the nuclear volume to
that of the cytoplasm is broadly invariant, reflecting the need
to balance nuclear and cytoplasmic processes. The correla-
tion was first stated between nuclear volume and cell (cyto-
plasm) volume, but, as nuclear volume depends on nuclear
DNA amount, the correlation also holds between nuclear
DNA amount and cell volume, although in a somewhat
relaxed manner. It is, of course important to realize that
there is a huge range in cell and nuclear size even within
the same organism due to cell differentiation (e.g., embryo
sac polar nuclei and nucellus nuclei are extremes in close
20 Genome Size and the Phenotype
Fig. 20.1 The positive relationship between genome size and guard cell
length. (a) Images of guard cells from four angiosperm species arranged in
increasing C-value (N.B. all images at the same magnification, scale bar
¼ 5 mm). (b) Graph plotting the relationship between log 2C DNA amount
and log guard cell size for 101 angiosperm species from 34 families.
(Images in (a) and graph shown in (b) are taken from Beaulieu et al. 2008)
proximity, the former decondensed and voluminous, the
latter condensed and small) so correlations between cell
size and genome size can only be made when comparing
the same cell type. So, for example, in animals, the size of
nucleate and even enucleate red blood cells show an impres-
sive correlation with genome size (Gregory 2005), while in
plants, guard cell size is clearly correlated with C-value
across a diverse sample of angiosperms (Beaulieu et al.
2008) (Fig. 20.1). In addition, it is important to note that
the best correlations are found among taxonomically close
relatives with different genome sizes, since in fairly distantly
related species, other developmental factors may come into
play and weaken the relationship. For instance, while
Bennett (1972) found a significant correlation between pol-
len size and genome size in 16 species of wind pollinated
grass species, Knight et al. (2010) were not able to confirm
this in a large scale analysis of 464 angiosperm species
belonging to 85 different families.
The clearest correlations between nuclear volume, C-
value and cell size are seen in meristematic cells that lack
conspicuous vacuoles. In differentiated plant cells additional
factors may obscure the correlation. For example, the pres-
ence of vacuoles makes quantification of the cytoplamic
volume difficult, while cell volume can also be influenced
by the particular stage in the cell cycle (replicated or
unreplicated, 2C or 4C), and by endopolyploidy (see
325
Maluszynska et al. 2013, this volume). In summary, the
size of differentiated organs often does not show a
clear relationship with genome size, so that spontaneous
polyploids are not always easy to recognize in the field.
One final point regarding the relationship between
nuclear volume, C-value and cell size is the observation
that, as genome size increases, the amount of (broadly
defined) heterochromatin also increases (Flavell 1980). As
heterochromatin tends to be more highly methylated and
condensed than euchromatin (Houben et al. 2003), the rela-
tionship between nuclear volume and genome size may not
be linear in species with genomes at the upper end of the
range of C-values encountered in plants.
20.2.2 Large Genomes Are Correlated
with Long Nuclear Cycles
It has long been appreciated that genome size and cell
division rate are linked, with the first studies being
conducted in plants by Van’t Hof and Sparrow (1963).
They studied six diploid angiosperm species growing at
23 C and noted a positive relationship between the duration
of mitosis and C-value. Since then there have been numerous
other studies although the results have not always been so
clear-cut. Thus, while some cell cycle studies suggested that
cell cycle length increased by 18–38% every time genome
size doubled (Van’t Hof 1965; Evans and Rees 1971; Evans
et al. 1972), other studies, such as those of Murı́n (1976),
pointed to a much more modest effect of C-value. For
example, while Vicia faba (2n ¼ 12) and V. sativa
(2n ¼ 24), differ seven-fold in C-value (Bennett and Leitch
2010), the cell cycle length for the larger genomed V. sativa
was only 1.27-fold longer. In part, such contrasting
observations may reflect the effect of numerous other factors
which can influence cell cycle length independently of
genome size (Grif et al. 2002). Indeed, Grif (2000) and
Grif et al. (2002) reported that some plants with different
genome sizes but grown at their own specific optimum
temperature (when the cell cycles in root tips are shortest)
had broadly the same cell cycle times. So, for example, the
ephemeral Arabidopsis thaliana (2n ¼ 2x ¼ 10; 0.16 pg,
1C; 22 C) and annual Avena sativa (2n ¼ 6x ¼ 42;
11.73–13.23 pg, 1C; 25 C) were reported to have identical
cell cycle times of 8.5 h despite a 78-fold and 26-fold differ-
ence in C-value and Cx-value, respectively. The observations
of Grif and colleagues were, however, based on 31 species
(22 genera) of different ploidy levels and diploidization
status, and some families were better represented than others.
In addition, Grif et al. (2002) noted that there could be
considerable differences in the cell cycle time for the
same species depending on the method used for analysis.
Notwithstanding these uncertainties, at 23 C plants with
326
Fig. 20.2 Relationship between DNA C-value (pg) and cell cycle time
(h) in root apical meristem cells of 110 diploid and polyploid
angiosperms. (Reproduced from Francis et al. 2008)
very large genomes such as Trillium rhombifolium (2n ¼ 6x
¼ 30; 111.5 pg, 1C) and Paris quadrifolia (2n ¼ 4x ¼ 20;
60.1 pg, 1C) were shown to have much longer cell cycles of
110 h and 56 h, respectively (Grif 2000; Grif et al. 2002).
In the most comprehensive overview to date, Francis
et al. (2008) reported a strong effect of genome size on cell
cycle time for 110 species, regardless of ploidy level and
life cycle (see Fig. 20.2). The strongest relationship was
observed in diploid, perennial monocots with the huge
genomes of Lilium and Trillium having disproportionately
long cell cycles. However, it should be noted that the outly-
ing huge genomes of Trillium and Lilium influenced the line
of the regression in favour of diploids, monocots and
perennials to the extent that the effect of ploidy may have
been obscured. Nevertheless, even if the outlying monocots
were excluded from the regression analysis, it was noted that
at a given C-value the variation in cell cycle times was
considerable, indicating the involvement of other factors
affecting cell cycle time.
The influence of polyploidy on cell cycle times is still
far from clear, but in part, this is likely to reflect where
in the diploidization/polyploidization cycle a particular
species falls (see Fawcett et al. 2013, this volume). In
neopolyploids cell cycles are not necessarily longer than
their corresponding diploids. Indeed, they may be shorter
as noted by Bennett and Smith who found that the duration
of meiosis in wheat was 42 h for diploids, 30 h for tetraploids
and 24 h for hexaploids despite the proportionately higher
DNA contents of polyploids (Bennett and Smith 1972). This
may arise because the ratio of inert versus genic DNA is
J. Greilhuber and I.J. Leitch
balanced. However, in older, more established polyploids
that have undergone considerable diploidization, often
accompanied by the elimination of non-informative DNA
and silencing of some informative DNA, the effect of poly-
ploidy on the relationship between C-values and cell cycle
length may become more obscure. Indeed, this may contrib-
ute to the lack of a clear effect of polyploidy on cell cycle
time in the large analysis by Francis et al. (2008) that
included polyploids at all stages of the diploidization/
polyploidization cycle.
It has been suggested that phylogeny may play a role in
determining the strength of the relationship between cell
cycle time and DNA amount. For example, Evans et al.
(1972) reported that although the slope of the relationship
between C-value and cell cycle time was similar between six
monocots and six eudicots (over the range of C-values stud-
ied), the cycle time for eudicots was approximately 4 h longer
than that of monocots of equivalent C-values, with the
increased duration primarily accounted for by a longer G1
phase of the cell cycle. They suggested that this may be due to
the denser chromatin packaging observed in the species
analysed (Evans et al. 1972). Nevertheless, in the larger
study of Francis et al. (2008) a similar, strong effect of
phylogeny was not so clear, although a marked increase in
cell cycle time in monocots with genomes larger than 25 pg
(1C) (i.e., Lilium and Trillium) was noted. It was suggested
that this may reflect the increased packaging of DNA into
heterochromatin in species with large genomes, reducing
access for the replication machinery and hence leading to a
longer DNA synthesis phase and hence longer cell cycle time.
One further factor that has been shown to influence cell
cycle times is the presence of B chromosomes, although data
are sparse. Ayonoadu and Rees (1968) showed that the
presence of B chromosomes increased the duration of the
mitotic cycle in Secale cereale while Evans et al. (1972)
reported that B chromosomes increased both the S-phase and
mitotic phase of the cell cycle in three grass species (Lolium
perenne, Zea mays and Secale cereale). However, to date the
mechanisms responsible are unclear. Evans et al. (1972)
proposed that both genotypic effects and/or the late replica-
tion of B chromosomes could play a role although they noted
that only Zea mays and Secale cereale were late replicating.
Overall it seems clear that although genome size can
considerably influence the length of the cell cycle and indeed
will set a minimum time, both genetic (e.g., genotypic
differences and variation is gene dosage owing to poly-
ploidy) and abiotic factors (e.g., temperature) can act to
increase its duration.
It is perhaps noteworthy that in contrast to eukaryotes, the
prokaryotic bacteria show a strong linear correlation between
genome size and gene number, but no significant correlation
between genome size and doubling time under laboratory
conditions, although there is considerable variation in growth
20 Genome Size and the Phenotype
rates between taxa (Mira et al. 2001). The reason may be that
bacteria do not contain significant amounts of accessory
DNA. This may be a consequence of the circular genome
they possess, which cannot exceed a certain size to avoid cell
division problems. In addition, recombination between direct
repeats within the circle may result in the formation of two
rings (depending on the resolution of the Holliday junction
(s)) and this may be lethal if the two circles fail to segregate
properly into daughter cells. In contrast, eukaryotes have
linear chromosomes which make the accumulation of acces-
sory DNA possible (Schubert 2011).
20.3 Hypotheses About the Biological
Significance of Genome Size Variation
Over the years there have been numerous attempts to try and
explain the observed correlation between genome size and
cell size and the biological significance of genome size
variation. Most of these theories can broadly be divided
into three contrasting hypotheses.
20.3.1 Accessory DNA is Mostly of Selfish Origin
and Represents Inert Genetic Junk
This hypothesis states that variation in genome size arises
purely from mutational events that lead to the differential
amplification of non-essential DNA (largely comprising
transposable elements (TE) and sometimes referred to as
parasitic, selfish or junk DNA). Species with larger genomes
thus represent those that are better able to tolerate the ‘self-
ish’ accumulation of DNA than those with smaller genomes.
Under such a scenario, the relationship between genome size
and cell size is considered to be purely coincidental rather
than biologically significant (e.g., Doolittle and Sapienza
1980; Orgel et al. 1980; Petrov 2002; Oliver et al. 2007).
The idea of parasitic genetic elements dates back to
Östergren (1945), who stated that B chromosomes did not
need to have a positive function for the organism in order to
exist, they just needed to be able to be self-sufficient, i.e., for
their reproduction, like a parasite and able to propagate
themselves. Given that mechanisms exist that can accumu-
late B chromosomes (e.g., preferential distribution into
gametes by non-disjunction, see Houben et al. 2013, this
volume), one could argue that this is sufficient to explain
their existence in a population without the need to invoke an
advantage for the organism. Comparable to B chromosomes,
TEs are also able to replicate and amplify within the genome
(Bennetzen 2005) and so perhaps no other explanation for
their existence need be sought. Nevertheless, it is possible to
envisage that the neutral accumulation of TEs during the
evolution of a species can only continue until developmental
327
constraints (e.g., through excessively long cell cycles or
heterochromatization) slow down or stop the reproduction
of the organism. In addition, it is now clear that molecular
mechanisms exist which are capable of eliminating DNA
(see review by Grover and Wendel 2010). These have been
shown to operate not only in diploids but also following
polyploid formation, leading to genome downsizing (see
below, and Fawcett et al. 2013, this volume). Such
observations suggest that while selective neutrality could
exist within certain limits, large increases in genome size
clearly can have certain deleterious effects, which makes a
predominantly neutral role of accessory DNA unlikely.
Indeed, such theories are also unable to explain why the
relationship between genome size and cell size persists fol-
lowing genome size reductions.
20.3.2 The Nucleotype Hypothesis
The correlations of genome size with cell size and cell cycle
time form the basis of most hypotheses that ascribe a
biological role to genome size, i.e., genome size variation
is conceived as being subject to selection rather than
representing a neutral character.
Although various researchers in the 1960s suggested that
the total mass of DNA played a role in influencing various
cell parameters (Commoner 1964; Martin 1966), it was
Bennett who coined the term ‘nucleotype’ to describe “that
condition of the nucleus that affects the phenotype indepen-
dently of the informational content of the DNA” (Bennett
1971, 1972). It should be noted that this definition includes
not only the total DNA mass, but also other possibly relevant
traits such as GC content (which can effect thermal stability,
mutation rate and cost of synthesis; see Šmarda and Bureš
2012, volume 1), and amount and arrangement of satellite
DNA (which can influence the extent to which DNA is
compacted into heterochromatin). Nevertheless, DNA
amount is considered the most important and is so far the
only nucleotypic parameter that has been studied in detail.
Bennett (1972) investigated the distribution of C-values
in herbaceous angiosperms and observed that the ranges of
C-values for each type of life style were different. He noted
that perennial herbs varied widely in C-values, whereas
annuals exhibited a much narrower range, with C-values
restricted to the lower end of the scale. In ephemeral species
(germinating and flowering within a few weeks, such as
Arabidopsis thaliana) only very small genomes were
found, with no species having a genome larger than 3.5 pg/
1C. Based on the observation that plants with small genomes
exhibit faster mitotic and meiotic cell cycles compared with
those possessing large genomes (see Sect. 20.2.2 above),
Bennett (1972) postulated a causal link between genome
size and life style, in which threshold effects were
328
considered to play an important role. He noted that, as
genome size increased, this was accompanied by an increase
in the minimum generation time (MGT—i.e., the shortest
time between germination and the production of the first
mature seed) and, at various points this resulted in an inescap-
able shift in developmental life style. Thus for a species to be
an ephemeral it had to possess a genome small enough to
enable a MGT of 7 weeks, whereas species with C-values
greater than the threshold value for a MGT of 52 weeks
(estimated to be about 25 pg /1C) were restricted to being
obligate perennials, in part because the time needed for the
required number of mitoses and meiosis is too long for an
annual lifestyle.
Bennett (1972) recognized that there were many limiting
factors which can act to slow down the MGT of a plant (e.g.,
availability of nutrients, temperature, genes) but they serve
only to increase the duration of the life cycle above a certain
minimum set by genome size. Thus while species with very
small genomes may be ephemeral, annual or perennial, as
genome size increases the number of life style options are
reduced until, above the c. 25 pg/1C threshold, species are
all obligate perennials.
At this point two complications of the nucleotype
hypothesis must be mentioned. First, it matters for cell
cycle length and thus for life style too, whether the genome
is diploid or polyploid. As noted above (Sect. 20.2.2), in
Triticum aestivum (hexaploid bread wheat), the meiotic
cycle was shown to be shorter than for tetraploid and
diploid wheats T. dicoccum and T. monococcum respec-
tively (Bennett and Smith 1972). Moreover, in maize,
artificial triploids and tetraploids were shown to have the
same cell cycle length as the diploid parent strain (Verma
and Lin 1979), and similar results were also reported for
diploid and autotetraploid cytotypes of Avena strigosa
(Yang and Dodson 1970). This absence of an increase in
cell cycle length is easily explained in part to be a conse-
quence of everything being multiplied in a polyploid—i.e.,
not only the DNA quantity but also the machinery to
replicate it, transcribe it into mRNA and translate it into
proteins. So, it seems to be the Cx-value, which influences
cell cycle time most strongly. However, in contrast, as far
as cell size is concerned, it is noted that the direct and
positive correlation between this character and DNA quan-
tity is based on the total DNA amount in the nucleus. So, it
is the C-value that determines the cell volume at least in
meristematic cells (N.B. as noted above, differentiated cells
have vacuoles, which introduce a strong element of varia-
tion in total cell volume and obscure the relationship
between cell size and DNA amount). If developmental
speed in terms of body size is adaptively important, the
best combination from the nucleotypic viewpoint would
seem to be a small Cx-value (fast cell division) plus poly-
ploidy (larger cells).
J. Greilhuber and I.J. Leitch
The second complication is that cell size and cell cycle
time can counteract each other in regard to developmental
speed, at least under certain circumstances (Price and
Bachmann 1976). When fast growth and attained final size
are critical (e.g., in understory plants), a larger cell size
(through larger genome) can perhaps promote growth better
than faster cell divisions (through smaller genome), because
the impact of increasing genome size is less on the cell cycle
than on the cell volume. This observation is based on early
studies which showed that at a constant ploidy level, a
doubling of genome size resulted in about a 1.18–1.38-fold
increase in cell cycle length (Van’t Hof 1965; Evans and
Rees 1971; Evans et al. 1972), but a two-fold increase in cell
volume (Price et al. 1973; Edwards and Endrizzi 1975).
Nevertheless, more data on this relationship are needed.
20.3.3 Nucleoskeletal DNA Hypothesis
In contrast to the nucleotype hypothesis outlined above,
Cavalier-Smith proposed that it was cell size which was
adaptive and under selection (Cavalier-Smith 1978, 2005).
Given that (1) the relationship between cell size and nuclear
size is much stronger than that between cell size and genome
size, and (2) that cell size is determined primarily via genetic
control rather than genome size, Cavalier-Smith (1978) put
forward the hypothesis that the role of DNA (including both
genic and accessory DNA) in a genome was primarily to act
as a nucleoskeleton which determined the nuclear volume
(both via DNA amount and the degree of packaging),
enabling the ratio between the nuclear and cytoplasmic
volume (¼ nucleo-cytoplasmic ratio) to be optimized. This
is considered essential to balance the overall rate of RNA
transcription and processing with that of protein synthesis to
allow balanced growth of actively dividing and growing
cells (as noted in Sect. 20.2.1).
With this hypothesis, the driving force is selection for an
optimal cell size; following the maxim “Modify your cell
size, the genome will follow!” For example, if a larger cell
size became adaptively favorable due to a change in selec-
tive forces, Cavalier-Smith envisaged that there would also
be positive selection for a corresponding increase in nuclear
volume and hence genome size, achieved primarily through
increases in the amount of non-coding DNA (although
modifications to the folding of DNA can also play a role in
determining nuclear volume).
Cavalier-Smith outlined many examples to support his
hypothesis (e.g., Cavalier-Smith 2005). One nice illustration
is provided by the miniature, enslaved nuclei known as
nucleomorphs that are found in two lineages of algae—the
cryptomonads and the chlorarachneans (Cavalier-Smith
2002). It is widely accepted that these two algal lineages
arose independently via secondary endosymbiosis whereby a
20 Genome Size and the Phenotype
eukaryotic alga (a green alga in the case of chlorarachneans
and a red alga in cryptomonads) was engulfed by a proto-
zoan (Green 2011). All that remains of the engulfed alga
today is the original plasma membrane, the chloroplast
(which provides photosynthate to the host cell), and its
nucleus (to enable the chloroplast to function). This enslaved
nucleus or nucleomorph is surrounded by a typical eukary-
otic nuclear membrane, contains chromosomes and
multiplies by normal cell division, and yet its genome has
undergone a remarkable reduction in size. Indeed, the
nucleomorphs have the smallest nuclear genomes so far
reported for any eukaryote, comprising just 551 kb for the
cryptophyte Guillardia theta (Douglas et al. 2001) and
373 kb for the chlorarachnean Bigelowiella natans (Gilson
et al. 2006).
Despite the independent origins of the nucleomorphs,
sequencing of their genomes has revealed similar genomic
architecture (e.g., genome divided into three chromosomes
c. 100–200 kb long, high gene density, similar proportions of
genomic components, and negligible amounts of non-coding
DNA) (Gilson et al. 2006). What is so intriguing is that the
extreme genome size reduction of the nucleomorph has
occurred within the same cell as the main (host) nucleus,
which is considerably larger. Indeed, the large range of cell
sizes observed in different species of cryptomonads scale
with the genome size of the main nucleus, exactly as
observed in other eukaryotes (Beaton and Cavalier-Smith
1999). Such an observation is consistent with the
nucleoskeletal hypothesis if one envisages that expansion
in the main nucleus has arisen through positive selection
acting on cell size that has been accompanied by an increase
in genome size via amplification of non-coding DNA to
provide the nucleoskeleton to balance the nucleocytoplasmic
ratio. The fact that selection can reduce genome size in one
nucleus (i.e., the nucleomorph) and alter genome size in the
main nucleus adaptively within the same cell is considered
to provide compelling evidence for a nucleoskeletal role of
DNA.
Overall, the distinction between the nucleotype and
nucleoskeletal hypotheses is rather subtle and is primarily
based on the target on which selection acts. In the nucleotype
hypothesis the cell size and/or cell cycle time is optimized
via selection for an appropriate genome size (Bennett 1972),
whereas in the nucleoskeletal hypothesis the target of selec-
tion is cell size, and genome size follows passively, because
a concomitantly larger or smaller nucleus allows accumula-
tion or requires deletion of skeletal DNA.
In reality both hypotheses probably apply during adaptive
evolutionary processes although the importance of each may
vary depending on a multitude of factors. In single celled
organisms such as bacteria, unicellular algae, protists, and in
organisms with blood cells (animals) it may well be that cell
size is most critical. For example, given that larger blood
329
cells have lower surface area to volume ratios and are there-
fore less efficient for gas exchange than smaller blood cells,
it is plausible that selection may act at the level of cell size
instead of genome size to optimize gas exchange to meet
metabolic demands (Gregory et al. 2009). On the other hand,
the cell size argument may be less clearly applicable in
land plants, on which Bennett primarily focused with his
nucleotype theory. Indeed, it is often difficult to predict
where the selective bottleneck is for a plant species. Is it
root growth rate, plant biomass, leaf area, reproductive rate,
generation time, pollen grain weight, pollen tube growth
rate, vessel diameter, stomata size and density, or seed weight
and reserve substance content? Certainly, investigations
into links between cell size and selection are often compli-
cated by the presence of vacuoles (which make estimations
of the cytoplasmic volume difficult) and endopolyploidy
(Maluszynska et al. 2013, this volume), which interferes
with genome size/cell size correlations as noted above.
Even for cell types which show reasonable correlations
between cell size and genome size (e.g., guard cell sizes:
Beaulieu et al. 2008, Fig. 20.1), taxon-specific factors as well
as evolutionary history are likely to play an important role in
determining how a particular lineage may respond to
prevailing selective forces.
20.3.4 Alternatives to the Nucleotype
and Nucleoskeletal Hypotheses
20.3.4.1 Reproduction Mode and Genome Size
Since an annual life history is preferentially associated with
inbreeding (Stebbins 1957; Ehrendorfer 1970; Barrett et al.
1997), it is possible that the correlation between low genome
size and annual life style noted by Bennett (1972, see
Sect. 20.3.2 above) is actually driven by an association
between reproductive mode and genome size, with
inbreeding species characterized by smaller genomes than
outbreeders. Indeed, several authors have addressed this
question.
Early studies reporting a correlation between genome size
and breeding system include those of Govindaraju and Cullis
(1991) who conducted a survey of 176 seed plants, and
Labani and Elkington (1987) who investigated Allium.
Both studies reported smaller genomes in selfing species.
However, no correction for phylogenetic relatedness was
made in either study. Indeed, the study of Govindaraju and
Cullis (1991) included 25 species of distantly related out-
breeding gymnosperms with large genomes (e.g., Pinus,
Picea, and Abies) and it is possible that they could have
influenced the correlations reported. Certainly the results
contradict another study by Rees and Jones (1967) who
reported the inverse correlation, with inbreeding Lolium
330
species characterized by genomes that were 30% larger than
their outbreeding relatives.
To test the relationship between genome size and breed-
ing system more fully Albach and Greilhuber recognized the
need to use phylogenetically-corrected statistical tests that
take evolutionary history into account (Albach and
Greilhuber 2004). This is because species do not represent
independent samples (as is assumed with most conventional
statistics methods). Indeed phylogenetic history has been
shown to be an important factor in explaining much of the
genome size variation encountered. Using phylogenetic
independent contrasts (Felsenstein 1985) and generalized
least squares approaches (Pagel 1997, 1999) Albach and
Greilhuber (2004) found better statistical support for an
association between low genome size with selfing than
with annuality in 42 Veronica species, with twelve selfing
species exhibiting significantly lower Cx-values than
outbreeders and three closely related selfing/outcrossing sis-
ter species pairs consistently showing the lower Cx-value in
the selfer.
Similarly in a phylogenetically-controlled comparison
between 14 taxonomically paired outcrossing and highly
selfing species comprising eight families including both
monocots and eudicots, Wright et al. (2008) found that
selfers had significantly smaller monoploid genome sizes
(i.e., removing the effect of polyploidy) than outcrossers.
To explain how inbreeding might affect genome size
evolution a number of mechanisms have been proposed.
For example, given the strong relationship between genome
size and amount of transposable elements (TEs) it is possible
that changes in the breeding system are accompanied by
changes in TE dynamics with inbreeding playing a role by
eliminating TEs more efficiently than outbreeders. Indeed
computer models have suggested that the smaller genome
sizes observed in some inbreeders arise due to a reduced
spread of TEs between individuals caused by the lack of
outbreeding (Wright and Schoen 1999; Morgan 2001), while
the ability of inbreeders to more effectively purge TE
insertions with deleterious recessive effects on fitness will
also lead to smaller genomes in inbreeders compared with
outbreeders (Wright et al. 2008).
Nevertheless, it has also been observed that the evolution
of self-fertilization may be associated with a large reduction
in the effective rate of recombination and a corresponding
decrease in effective population size (Wright et al. 2008). It
is therefore possible that the smaller effective population
sizes in inbreeders may counteract the effect of TE elimina-
tion noted above, due to the lowered efficiency of natural
selection to eliminate proliferating TEs (Lynch and Conery
2003). Under such a scenario, one might predict inbreeders
would have larger genomes than outbreeders.
In a much larger study analysing over 205 species of
angiosperms, Whitney et al. (2010) found only a very weak
positive correlation between genome size and breeding
J. Greilhuber and I.J. Leitch
system when phylogenetic history was taken into account,
suggesting that there is not a strong evolutionary association
between these two characters. Nevertheless, they noted the
discrepancy between their results and those of Albach and
Greilhuber (2004) and suggested it may be a consequence of
the different phylogenetic spread of the two datasets and the
impact of paleopolyploidy on genome size evolution.
Whereas Whitney et al. (2010) analysed 205 species from
across the seed plants, which included species at all stages of
the diploidization/polyploidization cycle, the study of
Albach and Greilhuber was restricted to a single angiosperm
genus where the impact of polyploidy was more controlled,
perhaps enabling the relationship between the reproductive
system and genome size to be more apparent. Clearly more
data are needed with analyses conducted at all taxonomic
levels to determine to what extent the taxonomic scale is
important in revealing the interaction between genome size
and reproductive mode.
Certainly these studies highlight the complexity of the
relationship between genome size and reproductive mode
and suggest that the importance of breeding system on
genome size may vary under different conditions. Indeed,
with annual inbreeders it is also possible, that in an a priori
small genome (such as in Veronica) the effect on genome
size of lethal TE insertions is stronger (as gene density is
higher) than in a larger genome (where gene density is lower
due to the larger amounts of repetitive DNA). In contrast, in
inbreeding taxa with a priori larger genomes the effect of TE
insertions on genome size and cell cycle length may be more
evident and selectively effective. The expectation would
thus be (1) in a higher taxon comprising a range of small
genomes, the inbreeders might be expected to have the
smaller genome sizes (as in Veronica), while (2) in a taxon
comprising a range of larger genomes, the annuals would
have the lower genome sizes. Given such a scenario, it is
perhaps plausible to consider that both mechanisms may be
operating within angiosperms.
One should also acknowledge that in animals, where
selfing is practically unknown, there is clear support for a
short life cycle/low genome size/small cell size correlation
(Gregory 2005).
20.3.4.2 Genome Size and Phosphate (P)
Availability
It has long been recognised that phosphate is an essential
macro-nutrient, playing a key role in many cellular
components such as nucleic acids (DNA and RNA), mem-
brane lipids and enzymes. Yet despite its abundance in the
environment it is not readily accessible to the plant and is
often present in such low amounts that it may be considered to
be a limiting nutrient for DNA biosynthesis and growth
(Raven et al. 2005; Nussaume et al. 2011). Indeed, some
plants have evolved specific mechanisms to cope with limit-
ing levels of P. For example, it has been observed that
20 Genome Size and the Phenotype
unicellular algae under chronic P-limitation have the capacity
to restructure their biochemical make-up by replacing P-rich
membrane lipids with sulphur- or nitrogen-containing lipids
(Van Mooy et al. 2009). There is also evidence that nutrient
limitation may directly affect the DNA composition of plant
genomes (Acquisti et al. 2009; Bragg and Wagner 2009).
Given these observations, it has been hypothesized that in
P-depleted soils there will be selection for species with
reduced genome sizes or metabolic demand for RNA as a
way to reduce the biochemical cost of synthesising DNA and/
or RNA, which are both phosphorous–rich molecules. The
hypothesis was initially based on the observation that very
small genomes were found in some carnivorous plants such as
Drosera which live in mineral-poor environments (Hanson
et al. 2001b) although since then very small genomes have
also been noted in other genera growing in nutrient-limited
environments such as Bixa orellana which occurs in the
mineral depleted soils of tropical America (Hanson et al.
2001a). Indeed, the smallest genome so far reported for a
plant is found in the carnivorous Genlisea aurea (Greilhuber
et al. 2006) which grows in tropical nutrient-poor
environments on the flat top of granite rocks. Nevertheless,
it should be noted that Genlisea is a genomically fast-evolving
genus (Jobson and Albert 2002; M€ uller et al. 2006), and so it
is possible that genome miniaturization may have arisen as a
result of intrinsic genomic factors as a consequence of the
carnivorous mode of nutrition rather than due to external
selection pressures (Albert et al. 2010).
As an extension of the hypothesis it has also been
suggested that polyploidy might be counter-selected under
strong P-limitation (Leitch and Bennett 2004; Leitch and
Leitch 2008), a hypothesis that finds indirect support from
studies indicating that increases in plant ploidy level are
often associated with evolutionary reductions in genome
size (Leitch and Bennett 2004). Nevertheless, it is clear
that further work is necessary to determine the extent to
which P-limitations play a role in the establishment and
evolution of polyploids in comparison with related diploids.
Indeed, cytological studies of plants living in environments
with low P-levels such as bogs and heathlands or in some of
the highly nutrient-depleted soils of Australia (Beadle 1962)
may be particularly informative to determine to what extent
P-levels influence the incidence of polyploidy.
20.4 Examples and Explanations
20.4.1 Cell Size, Genome Size and Sperm
Mobility in Bryophytes
Genome sizes in bryophytes are much less variable than in
tracheophytes (i.e., c. 38-fold in liverworts and 12-fold in
mosses, with no data for hornworts—see Leitch and Leitch
331
2013, this volume). To explain this limited variation in
genome size, Renzaglia et al. (1995) proposed that it was
because of the nucleotypic effect of genome size on sperm
size and mass. This arises because the nucleus contributes
most of the cell mass in sperm cells, thus there is a reason-
able correlation between sperm cell size and C-value. Since
the motility of sperm depends on their size and hence the C-
value, it might be envisaged that there would be selection
against too much DNA as this would result in larger sperm
making them less mobile and hence less efficient at effecting
fertilization. Support for the hypothesis comes from the
observation that lycophytes with biflagellate sperm are also
characterized by very small genomes (e.g., Selaginella)
whereas lycophytes and gymnosperm groups with
multiflagellate sperm (e.g., Isoetes and Phyloglossum in
lycophytes and cycads and Ginkgo in the gymnosperms)
have larger genomes as might be expected as they will be
less constrained in this regard (see Leitch and Leitch 2013;
this volume).
20.4.2 The Impact of Genome Size
on Phenology
In 1982 Grime and Mowforth put forward an interesting
hypothesis concerning the ecological significance of genome
size on plant phenology and relating it to the impact of
genome size on cell cycle length and cell size (Grime and
Mowforth 1982). They observed that in a woodland commu-
nity of flowering plants in the British Isles, the first species to
flower in the year (e.g., snake’s head fritillary—Fritillaria
meleagris and lesser celandine—Ranunculus ficaria) were
characterized by possessing the largest genomes of the spe-
cies analysed while as the year progressed, the later
flowering species had progressively smaller genomes. The
explanation offered by Grime and Mowforth (1982) was
based on the relationships between genome size and cell
cycle time and between genome size and cell size (see
Sect. 20.2). They hypothesized that the plants emerging
and flowering very early in the spring had already preformed
their organs during the warm period of the preceding season
via cell division but without cell expansion. In the following
year, with the availability of water after the frost period, a
fast expansion of the cells by water uptake could then take
place, and given that cell expansion is temperature-
independent (unlike cell division), this enabled such plants
to grow through cell expansion at a time of year when it was
too cold for growth via cell division. Grime (1983) also
observed that the rate of leaf expansion was faster for species
with larger genomes, thus plants with larger genomes would
be able to expand their leaves and hence ‘grow’ faster than
the smaller genomed species, providing an additional advan-
tage of larger genomes for such early growing plants. Those
332
plants flowering later, which did not separate cell division
from cell expansion, were limited to growing only once
favorable temperatures were reached to enable cell division
and were characterized by smaller genomes and hence faster
cell cycles. Greilhuber (1995) pointed to the analogous situ-
ation in summer-dry habitats, where bulbous and rhizoma-
tous plants with large genomes also thrive.
The same correlation, i.e., increasingly smaller genome
sizes in species flowering later in the year, was also noted by
Labani and Elkington (1987) in Allium and this was con-
firmed by Baranyi and Greilhuber (1999) for diploids. How-
ever, the latter authors failed to confirm the correlation when
polyploid Allium species were included in the analysis.
These results suggest that it is the Cx-value rather than the
C-value that matters in this relationship. Although more
recently Ohri and Pistrick (2001) failed to find any correla-
tion in yet another extensive analysis of genome size and
flowering time in Allium, it is currently unclear whether this
was due to technical or biological factors. For example, Ohri
and Pistrick used the static one-wavelength Feulgen photom-
etry method to estimate genome size in Allium and this has
been reported to give rise to problematic data (see
Greilhuber et al. 2007) and they also did not adjust for ploidy
level although their sample included many polyploids. It is
clear that further studies using flow cytometry for genome
size estimations not only in Allium but other genera as well,
together with adjustments for ploidy and the inclusion of
phylogenetic data will be necessary to determine the full
extent to which genome size plays a role in influencing
plant phenology.
20.4.3 The Relationship Between Genome Size
and Weediness
Weeds are plants invading open arable land and many of them
have characteristics of r-strategists, i.e., fast reproduction and
development, the production of many propagules, and an
annual life style being the most common. Given the
nucleotypic correlations between genome size (1C and 1Cx-
value) and rapid development one might predict that weeds
would be characterized by small genomes. To address this
question, Bennett et al. (1998) analysed 156 weed species
(comprising both British garden weeds and species
recognized as important weeds on a global scale) and com-
pared them to 2,685 other non-weed species. They observed
that (1) weeds were characterized by a narrower range of
genome sizes, limited to the bottom 20% of the range encoun-
tered in angiosperms as a whole, (2) the mean C-values and
Cx-values were significantly lower in weeds than in the non-
weed sample, (3) no weed was observed to have a 1C-value
greater than 25 pg and (4) the most aggressive weeds were
characterized by the smallest genomes (e.g., water lettuce,
Pistia stratiotes 1C ¼ 0.3 pg). Further analysis showed that
J. Greilhuber and I.J. Leitch
the probability of being a weed decreased with increasing 1C-
and 1Cx-values up until the threshold value of 1C ¼ 25 pg
was reached, and that the weeds with the largest genomes
were predominantly polyploid, in agreement with the obser-
vation that cell cycle times in polyploids are often shorter
compared to diploids of an equivalent 1C-value (see
Sect. 20.2.2 above), although the greater genetic diversity
observed in allopolyploids may be an additional factor under
selection in weeds. Overall, this study supports the prediction
that weeds are characterized by small genomes although it
seems likely that selection is acting on correlated factors such
as (1) rapid development, (2) fast growth and (3) production
of many small and light seeds that are easily dispersible, and it
is these evolutionary forces that may be shaping and
constraining genome size in weeds.
20.4.4 Genome Size and Invasiveness
Like weediness, to be a successful invasive requires pread-
aptation or fast adaptation to the new, usually disturbed
environments, in which competitive superiority regarding
speed of growth is a priority. Once again, cell size and cell
cycle time and associated parameters such as seed weight
and number, are considered to be important traits to be a
successful invasive, and given the link between genome size
and these characters, a small genome size has been
suggested to be an important prerequisite for plant invasive-
ness. Indeed, Rejmánek (1996, 2000) listed a small genome
size as one of the eight most important factors contributing
to the invasive success of a species, and several studies at the
generic level (including the angiosperms Artemisia (Garcia
et al. 2008), Briza (Rejmánek 1996), and gymnosperm Pinus
(Grotkopp et al. 2004)) and species level (e.g., Phalaris
arundinacea, Lavergne et al. 2010) have provided support
for this.
In a broader analysis, Kubešová et al. (2010) conducted
a flow cytometry-based genome size comparison between
alien plant species (including both naturalized and invasive
species) and their native counterparts in the flora of the Czech
Republic. In total, the study analysed 93 species comprising
both native and alien species from 70 genera and 32 families
and compared their genome sizes with those of congeneric
and confamilial non-invading taxa worldwide (using data
from the Plant DNA C-values Database). Like the more
focused studies mentioned above, this broad scale analysis
found that naturalized and invasive plants had significantly
smaller genomes (C-values and Cx-values) than their non-
invading relatives at both the generic and familial level and
indeed, no naturalized species with large genomes were
seen. The species with the highest C-value was Rudbeckia
laciniata, an octoploid with 1C ¼ 15.27 pg and 1Cx ¼ 3.82
pg. A comparative analysis of 401 species of the Californian
flora by Knight and Ackerly (2002) also found that the
20 Genome Size and the Phenotype
non-native species had significantly smaller genomes than
the native species, while an absence of large genomes and
overrepresentation of species with small to very small
genomes was observed in an analysis of 238 endemics from
the oceanic Macaronesian Islands compared with non-
Macaronesian relatives (Suda et al. 2005) as well as in a
survey of two Pacific archipelagos—Hawaii and Marquesas
Islands (Kapralov and Filatov 2011). In addition, a survey of
C-values for 3,676 angiosperms by Chen et al. (2010)
suggested that both Cx- and C-values had significant effects
on plant invasiveness with the exception of trees and species
belonging to Fabaceae. Taken together the results do suggest
that genome sizes may well be constrained in colonizing/
invading plant groups.
Evidence that smaller genomes may indeed be under
selection in invasive species comes from a study by Lavergne
et al. (2010). They analysed 90 genotypes of the reed canopy
grass (Phalaris arundinaceae) from N. America and Europe,
and observed that the genome size was significantly smaller
in the invasive genotypes from N. America compared with
the native European genotypes. By modelling the genome
size data within a phylogenetic framework they concluded
that the smaller genomes observed in the N. American
genotypes had been maintained by natural selection during
the invasive process. The increased invasive potential was
considered, in part, to be due to the more rapid early growth
observed in the invasive genotypes arising as a result of the
reduction in genome size. Nevertheless, it should be pointed
out that a survey of genome sizes in 92 species of Australian
Acacia comprising both invasive versus non-invasive species
failed to find any link between genome size and invasiveness
(Gallagher et al. 2011), although such a result was considered
to be most likely due to the very small DNA values of Acacia
species studied. Instead it was suggested that other traits,
such as plant height and range size, which were shown to
be significantly different between invasive and native spe-
cies, play a more important role in contributing to the inva-
sive success of some Acacias than genome size.
Overall, it seems clear that more comparative data are
needed to determine the extent to which a decrease in genome
size is a general evolutionary mechanism leading to the evo-
lution of more invasive species. Recent studies have shown
that polyploidy (and hence genome size increase, at least
initially) also plays a significant role in determining the inva-
siveness of plants, highlighting the complexity of interactions
influencing the ecological traits of a plant (Chen et al. 2010;
Pandit et al. 2011; te Beest et al. 2012).
20.4.5 Genome Size and Climate
Studies of genome size have often looked for correlations
with various climatic parameters. Of these, many have
reported relationships between genome size and elevation
above sea level (i.e., altitude) or geographical latitude
333
because they are considered to be associated with parameters
such as temperature, precipitation and length of growing
season. However, a comparison of results between different
studies has shown that the directions of the relationships are
not always consistent. For example, Knight et al. (2005)
noted that out of 18 studies which looked for a correlation
between genome size and latitude, five reported the relation-
ship to be positive, seven found a negative relationship and
six failed to find any relationship at all. Similar levels of
inconsistencies were also found in studies examining the
relationship between genome size and altitude. Given that
altitude and latitude are obviously related to climatic factors
but not always in the same way, it is perhaps not surprising,
that different studies have found different trends. A latitudi-
nal gradient can go from dry to moist or from mesic to (ant)
arctic, in the same way as a gradient from low to high
elevations. Indeed, it may well be that temperature and
precipitation are the more biologically relevant parameters
on which selection for genome size acts and these do not
vary linearly with altitude or latitude.
Another explanation for the rather confusing picture is that
the majority of studies failed to take phylogenetic history into
account in their analysis, a point noted by Bancheva and
Greilhuber (2006) in their study of Centaurea. Here, apparent
correlations of genome size with altitude, temperature and
precipitation disappeared when phylogenetically closely
related species were examined. These observations led to
the suggestion that the relationships were pseudo-correlations.
An alternative reason for the inconsistent relationships
could arise from technological problems. For example, a
previously claimed negative correlation between genome
size and latitude for Glycine max cultivars reported by
Graham et al. (1994) could not be confirmed by Greilhuber
and Obermayer (1997). The discrepancy between the two
studies was attributed to problems of using external
standardization for genome size measurements by Graham
et al. (1994). Such an approach has since been shown to lead
to serious errors (Greilhuber 1998, 2005) and best practice
methods of genome size estimation now emphasize the need
to always use internal standardization when estimating
genome size (Greilhuber et al. 2007).
On a broader scale, Knight and Ackerly (2002) suggested
that the lack of clear relationships between genome size and
various climatic variables could have arisen because many of
the studies were too narrow in their focus and did not represent
the full ecological range of a species (e.g., they did not encom-
pass the full range of latitudes from the tropics to the poles,
or altitudes from sea level to mountain tops). In addition,
they also recognized that the relationships being studied may
not be linear and so the application of linear regression
analysis may be inappropriate. For example, they noted the
study of Rayburn and Auger (1990) who found the relation-
ship between genome size and altitude in maize (Zea mays)
was non-linear. From an examination of 23 populations
growing at different altitudes, Rayburn and Auger (1990)
334
reported that those growing at sea level and high altitudes had
smaller genomes than those growing at intermediate
elevations. Similarly, for latitude, Bennett (1987) observed
that while genome size of a range of crop species initially
increased with increasing latitude, over a certain latitude
threshold, species with large genome sizes were progres-
sively excluded, presumably due to selection against species
with large genome sizes as the climate became harsher and
growing seasons shorter. To overcome these difficulties,
Knight and Ackerly (2002) decided to take a novel approach
by using quantile regression analysis to analyse genome size
diversity in relation to various climatic variables in 401 species
across broad environmental gradients of the Californian flora.
Their studies showed the relationship between genome size
and various environmental parameters was certainly not lin-
ear. Thus while species with small genomes were observed
across the range of temperatures and precipitation that they
encountered (and thus were poorly correlated with genome
size), as genome size increased, the correlations became
increasingly significant with large genomed species being
excluded from the extreme environments with shorter growing
seasons (i.e., low or high maximum July temperatures or
reduced annual precipitation).
Given these observations one might expect alpine species
which experience short growing periods to be characterized
by smaller genomes than relatives growing in non-alpine
habitats, and yet many have larger genomes. For example,
in the study of Veronica, Albach and Greilhuber (2004) used
phylogenetic independent contrasts (to take phylogenetic
history into account), and showed that alpine species had
significantly larger 1Cx-values than non-alpine ones. How-
ever, a similar analysis based on 1C-values was not signifi-
cant illustrating the point that for Veronica at least it is the
cell cycle relevant Cx-values that may be under selection in
these habitats. In other species it has been suggested that
large genomes may be an advantage in alpine habitats given
the observed positive relationship between genome size and
frost resistance (MacGillivray and Grime 1995). In addition,
it has been suggested that larger genomes may be
more readily tolerated in alpine species because the soils
are generally more phosphate-rich at higher altitudes
(K€orner 1989) and thus may not be under such strong selec-
tion to limit genome size in order to conserve phosphate
which is often in limiting supply in other habitats (see
Sect. 20.3.4.2). It should however, be noted that Bennett
(1987) argued against large genomes in alpine habitats due
to the observed positive correlation between genome size
and UV damage (Sparrow and Miksche 1961) and the fact
that UV levels in alpine habitats are often higher than at
lower elevations.
J. Greilhuber and I.J. Leitch
20.4.5.1 Genome Size and the Impact of Climate
Change
Given the observed complexity of interactions between
genome size and different climate variables, the question
of how plants may respond to climate change is likely to
be complex. Yet there are a number of additional studies that
are worth mentioning in this respect. For example, Jasienski
and Bazzaz (1995) investigated the effect of elevated levels
of CO2 on growth in annual grasses and observed that the
extent of the growth response was positively correlated with
genome size. In contrast, Grime (1996) reported that under
elevated temperatures plants with small genomes showed a
greater enhancement of growth compared with species with
larger genomes, with the implication that under a scenario of
global warming species with small genomes may expand
their range more successfully in certain areas (e.g., temper-
ate floras). Nevertheless, this predicted expansion could be
held in check in certain areas given the findings of yet
another study by MacGillivray and Grime (1995) showing
that such potentially responsive species with small genomes
are more sensitive to low temperatures and freezing than
those with larger genomes (as noted above for alpine spe-
cies). Thus the potential expansion of species with small
genomes under a warming climate would be expected to be
reduced in temperate areas experiencing occasional late
frosts.
It is clear even from these rather few and limited studies,
that predictions of how plants will respond to climate change
are fraught with difficulties, yet they do emphasize that
genome size does play a role and should be incorporated as
a character into models which aim to understand how changes
in climate will influence plant distributions in the future.
20.4.6 Genome Size and Pollution
As the world becomes increasingly polluted through human
activity, some researchers have questioned whether genome
size has any impact on plant survival in polluted
environments. Two notable studies are those of Temsch
et al. (2010) and Vidic et al. (2009) who used different
approaches to investigate a heavy-metal-polluted area in
Žerjav, in northern Slovenia known as Dolina Smrty. This
“Death Valley” provides an unintended but ideal experimen-
tal environment to investigate the question as the soil is
heavily polluted with lead from a disused metal smelter
and there is a strong gradient of lead pollution with
concentrations ranging from 30 g of lead per kg of soil at
the site closest to the smelter to 0 g/kg soil at a control site.
The studies involved surveying the species diversity and
genome sizes of all herbaceous dicots growing in five
20 Genome Size and the Phenotype
Fig. 20.3 Correlation between
concentration of lead in the soil
and 1C-values expressed as a
percentage of species with large
genomes (diamonds) and the
probability of finding by chance
that number or fewer large
genomes (circles) at the five test
plots of Dolina Smrty (Žerjav,
Slovenia). (Reproduced from
Temsch et al. 2010)
study plots and the findings were dramatic. Not only was the
number of species progressively reduced with increasing
pollution, but the genome sizes of the surviving species
were, on average, progressively smaller. The results suggest
that as the lead concentration in the soil increases, species
with larger genomes are dying out, or are being excluded,
compared to those with smaller genomes (Fig. 20.3).
Given that significant mitotic and meiotic disturbances
through genotoxic stress have been observed in species with
larger genomes from this area (Druškovič 1984), this may
explain in part, why species with larger genomes are increas-
ingly excluded as the level of lead pollution rises. Such a
correlated sensitivity of genome size with genotoxic stress
finds a parallel in the long known higher radiation sensitivity
of large genomes observed by Sparrow and Miksche (1961)
and may arise because larger genomes have larger and
denser nuclei, and thus provide a better target for damage.
Similar studies for related pollutants are clearly necessary to
determine to what extent such findings may be generalized
across the range of pollutants being pumped into the soil
through human activity.
20.4.7 The ‘Large Genome Constraint
Hypothesis’
From the above discussions, it is clear that the impact of
genome size on an organism is complex and, in part, is
determined by genome size itself, with the consequences
often becoming increasingly significant, and options available
being more limited as genome size rises. For example, as
Bennett (1972) noted, species with small genomes may
adopt any life cycle strategy (i.e., they may be ephemeral,
annual or perennial) while those with large genomes are
335
restricted to being obligate perennials (see Sect. 20.3.2).
This is a clear nucleotypic effect of genome size because
even if a species with a large genome contained all the genetic
code necessary to be, for example, an ephemeral, the long
duration of mitosis and meiosis as a consequence of its
genome size would prevent it from completing a sufficient
number of cell divisions to grow fast enough to be an ephem-
eral. Other observations discussed above also suggest that
having a large genome restricts the options available to the
plant, e.g., restriction to more mesic habitats (Knight and
Ackerly 2002), exclusion from being a weed or invasive
(Bennett et al. 1998; Suda et al. 2005; Kapralov and Filatov
2011), exclusion from living in a heavily polluted soil (Vidic
et al. 2009; Temsch et al. 2010). Studies such as these, together
with the observation that most angiosperms have small
genomes (see Leitch and Leitch 2013, this volume) despite
the prevalence of mechanisms that have the potential to gen-
erate large genomes (e.g., polyploidy and retrotransposon
amplification), suggest that there may indeed be selection
against large genomes. Based on observations such as these,
Knight et al. (2005) proposed the ‘Large Genome Constraint
Hypothesis’, suggesting that species with large genomes do
pay a cost and may, under certain circumstances, be selected
against. In support of their hypothesis they noted how it was
often necessary to adopt quantile regression analysis to tease
out the nature of the relationships in such non-linear systems
where the correlations only become significant at the upper
end of the range of genome sizes (as noted in their climatic
study—see Sect. 20.4.5).
20.4.7.1 The Large Genome Size Constraint
on Speciation Rates and Extinction
Two studies have been conducted to test whether the posses-
sion of a large genome constrains the ability of a genus to
336
speciate. Vinogradov (2003) found a negative correlation
between the mean genome size of a genus and the number of
species in a genus using Spearman’s rank correlation analysis,
although the correlation was weak (r ¼ -0.11, p < 0.001).
While Knight et al. (2005) also found a weak but significant
relationship using slightly different sample data and
Spearman’s rank correlation analysis, they also tested quantile
regression analysis to explore the nature of the relationship.
While they found no significant relationship between genome
size and diversification rates for species with small genomes,
presumably due to the myriad of factors which can influence
diversification rates, they did find an increasingly negative
trend for larger genomes providing support for the hypothesis
that genera with large mean genome sizes are less species rich.
Nevertheless, they recognized there were several potential
sources of errors in their analysis (e.g., poor representation of
species in some genera, the use of mean genome sizes to
represent a genus, different evolutionary ages of genera
analysed) and concluded that the evidence for a role of genome
size in species diversity was, at best, tentative. Indeed, it is also
noted that neither analysis was conducted within a phylogenetic
framework that could further confound the results.
A recent review by Kraaijeveld (2010) collated studies
across eukaryotes (including vertebrates, anamniotes,
amniotes, amphibians, reptiles, eutherians and fishes)
which had investigated whether there was any relationship
between genome size and species diversification in different
groups. He concluded that the general pattern seemed to be
one in which a higher diversification rate was generally
found in taxa with smaller genomes with the only exception
being the fishes where a positive relationship was found in
all subgroups except pufferfish. Since species diversity
represents the product of both speciation and extinction,
Kraaijeveld (2010) noted that the absence of species-rich
groups in taxa with large genomes could arise either due to
constrained speciation rate, or increased extinction rate or
both, although distinguishing between these possibilities is
currently not possible using available techniques.
In plants there is currently only one comprehensive study
that has been conducted looking at the impact of genome
size on extinction (Vinogradov 2003). Using data for 3,036
species with both genome size data and information on
threat of extinction taken from the United Nations World
Conservation Monitoring Centre Database, Vinogradov
showed that diploid species with large genomes were signif-
icantly more likely to be listed as rare or endangered com-
pared with those with small genomes, and the results were
independent of life history type or polyploidy. Although he
did not use approaches such as phylogenetic independent
contrasts he did also conduct analyses within families that
overcame, to some extent, complications arising from phy-
logenetic issues. However, given the importance of phylog-
eny in determining the distribution of genome sizes across
J. Greilhuber and I.J. Leitch
angiosperms (as previously noted), repeating the analysis
using phylogenetically-sensitive approaches is highly
recommended, especially since there are now considerably
more genome size data available and a greater understanding
of phylogenetic relationships across angiosperms.
Given the relationships observed between genome size
and many nuclear, cellular and phenotypic characters per-
haps one should not be so surprised by the suggestion that
species with large genomes may be at greater risk of extinc-
tion. As discussed above, the possession of a large genome
in many cases does constrain the potential of a species and
hence, in a rapidly changing world the more restricted
opportunities available may prevent large genomed species
from adapting fast enough and hence may be more likely to
be threatened with extinction.
20.5 Intrapopulational and Intraspecific
Variation of Genome Size: Adaptively
Important?
Over the many years of genome size research, scientists have
looked for evidence of intraspecific and intrapopulation var-
iation in C-values and there are now over 200 papers exam-
ining more than 80 genera which have addressed this
question (Šmarda and Bureš 2010). If such variation can be
reliably demonstrated, one might hope to find the starting
point of the large interspecific variation observed across
plants, and eventually to determine if there are ecological
correlates to explain why such variation might arise. Indeed,
if correlates could be found, then a function (selective role)
of genome size at the population level and above (within a
species) could be proven.
Despite the numerous investigations that have been made,
based on the increased understanding of the strengths and
weaknesses of the methods used to estimate genome sizes,
it has become clear that many of the Feulgen densitometry-
based results should be viewed with caution due to
technological shortcomings (unavoidable or otherwise, see
Greilhuber 1998, 2005, 2008). Nevertheless, some of the
early research looking for the presence of intraspecific varia-
tion was conducted with great care, such as the work of Price
and co-workers on species belonging to Microseridinae, a
N. American tribe within Asteraceae. In one such study,
they estimated genome sizes for 222 plants of Microseris
douglasii from 24 geographically, ecologically and morpho-
logically diverse populations in California and reported up to
1.20-fold variation in DNA amount among individuals within
populations and 1.14-fold range in mean DNA values
between populations. They also observed differences in
DNA amount in the same populations in different years
(Price et al. 1981). Overall the results suggested that there
was an ecological component to the DNA variation. Plants
20 Genome Size and the Phenotype
with larger genomes were limited to clay soils with a higher
moisture content than plants with smaller genomes, which
typically occupied soils of lower moisture content. Neverthe-
less, when a further analysis of 210 plants from 10
populations was conducted in subsequent years the expected
confirmation of observing higher DNA contents from
individuals and populations experiencing increased soil mois-
ture did not hold, highlighting the complexity of the relation-
ship between genome size and environmental parameters
(Price et al. 1986). To date, the findings of Price and
colleagues have not been confirmed using flow cytometry or
investigated with molecular tools. It is therefore unresolved
whether there is genuine intraspecific variation in Microseris
or whether the results are a technological artefact arising from
variable amounts of cytosolic inhibitors which can interfere
with the quantitative staining of DNA and lead to
pseudo-intraspecific variation, as observed for example in
sunflowers (Price et al. 2000) and coffee (Noirot et al. 2000;
Noirot et al. 2003).
Other studies reporting extensive intraspecific variation
from this era include the work of Mowforth and Grime
(1989) who noted considerable intrapopulational variation
in the grass Poa annua, with values between individuals
ranging 1.8-fold. Chromosome counts confirmed that all
individuals had the same number of 2n ¼ 28, thereby
eliminating the possibility that variation had arisen from
the presence of B chromosomes, aneuploidy or polyploidy.
In addition, the variation was shown to be positively
correlated with cell size and negatively associated with
variation in seedling growth rate. The suggestion that there
may be selection for different genome sizes attuned to dif-
ferent temporal and climatic niches was proposed to explain
the variation (Mowforth and Grime 1989). As for Microseris
douglasii, there has been no further work on this species and
so the results remain unconfirmed. Nevertheless, it seems a
perfect candidate to reinvestigate, especially given that
many of the reports of intraspecific variation arising during
this period have since been reinvestigated and shown to be
unreliable such as Dasypyrum villosum, Glycine max,
Arachis hypogaea, Collinsia verna (reviewed by Greilhuber
1998, 2005).
In recent years it has become clear that even with care-
fully conducted experiments, Feulgen microdensitometry is
no longer considered to be a suitable technique to detect
intraspecific variation, given its limitations in the speed and
number of samples that can be analysed. Instead, the increas-
ing refinement, accuracy and sensitivity of flow cytometry
(assuming best practice methods are followed) means that it
has now become the method of choice for detecting intra-
specific variation. Flow cytometry is also considerably faster
than Feulgen-based approaches enabling the rapid analysis
of large numbers of nuclei from many individuals. Best
practice techniques for convincingly demonstrating the
337
presence of intraspecific variation have recently been
outlined by Šmarda and Bureš (2010) and include: (1) the
presence of double peaks in the flow histogram when two
individuals differing in genome size are co-chopped and run
as a single sample in the flow cytometer, (2) the consistent
use of an internal standard for all measurements, (3)
demonstrating that the variation is reproducible by repeating
the measurements on different days, using different
machines and/or different fluorochromes, and (4) testing
for the presence of cytosolic inhibitors which have the
potential to interfere with the quantitative staining of DNA
by the flurochrome. Such developments have now opened up
the possibility of investigating the true extent of intraspecific
variation for the first time. Indeed, there are now an increas-
ing number of carefully conducted studies which have
followed these best practice approaches including the dem-
onstration of double peaks indicative of intraspecific varia-
tion (e.g., Pecinka et al. 2006; Šmarda 2006; Šmarda and
Bureš 2006; Walker et al. 2006; Leong-Škornickovà et al.
2007; Sch€onswetter et al. 2007; Suda et al. 2007; Achigan-
Dako et al. 2008).
Below several studies are outlined which have sought to
document the extent of intraspecific variation in genome size
diversity at various spatial and temporal scales and to link
this with various environmental factors to provide greater
understanding of the forces which might be driving the very
first incipient genome size changes which could ultimately
lead to the extensive genome size diversity observed across
plants.
20.5.1 Evolution Canyon
An area that has been extensively studied in terms of
investigating intraspecific variation in genome size is Evo-
lution Canyon I, Lower Nahal Oren, Mount Carmel, in
northern Israel (Nevo 2012). The 400 m wide canyon,
which dates back to the Plio-Pleistocene era (Nevo et al.
1999), is orientated in an east-west direction and thus
presents north and south facing slopes. Both slopes share
common geologies and macroclimates but have strongly
contrasting microclimates differing in the amount of solar
irradiation and aridity. The savannah-like south facing slope
(SFS) receives 200–800% more irradiation and is more arid
(‘Africa-toned’) than the forested north facing slope (NFS)
which is wetter (‘Europe-toned’). Consequently species on
the NFS are considered to be under shade stress whereas
those on the SFS are under drought stress (Nevo 2012).
Given these steep ecological gradients encountered in Evo-
lution Canyon I a number of studies have looked to see
whether there is any evidence of intraspecific variation in
genome size which can be linked with environmental
factors.
338
20.5.1.1 Hordeum spontaneum
Of special relevance are the studies in Hordeum species,
especially those of H. spontaneum, by Schulman and
associates, because here genome size, transposable element
(TE) variation, and ecology have been considered together.
Hordeum spontaneum (2n ¼ 14) is the wild progenitor of
barley, H. vulgare, and previous studies have demonstrated
the existence of intraspecific variation of up to 1.13-fold in
accessions collected from Israel (Kankanp€a€a et al. 1996;
Turpeinen et al. 1999). Like other Hordeum species, H.
spontaneum has been shown to contain a transcriptionally-
active TE known as BARE-1. This is a LTR-retrotransposon
belonging to the copia family, and is c. 8,932 bp in length
(Manninen and Schulman 1993) or 12,088 bp if a 3.14 kb
insertion in the 30 long terminal repeat is present (Suoniemi
et al. 1996a).
In a study of BARE-1 in different Hordeum species,
Vicient et al. (1999) analysed nine different accessions of
H. spontaneum collected from across Israel and showed not
only that there was intraspecific variation in genome size
between them (1C ¼ 4.14–4.68 pg) but that this was
correlated with the contribution of BARE-1 to the genome,
which ranged from 2.8% to 5.8%. Further they noted that the
genome size appeared to be higher in accessions collected
from desert areas, suggesting a link between genome size,
BARE-1 copy number and aridity.
To determine whether such a link was also found on the
microclimatic scale, Kalendar et al. (2000) conducted a
similar analysis to that of Vicient et al. at Evolution Canyon
I by sampling individual plants of H. spontaneum at six sites
across a 300 m transect which traversed the NFS and SFS
and differed in height. The study certainly showed that there
was considerable variation in the number of full length
BARE-1 elements, with copy numbers ranging between 8.3
and 22.1  103 copies per genome, comprising between
1.8% and 4.7% of the genome. In addition, they found
differences in the number of ‘solo long terminal repeats’
(¼ solo LTRs) between the sites. Solo LTRs represent
‘footprints’ of BARE-1 elements that have undergone intra-
element or possibly intra-chromosomal recombination
between the LTRs of the BARE-1 element resulting in the
elimination of the internal portion of BARE-1.
What was most noticeable was the correlation between
environmental factors and the copy number of either full
length BARE-1 elements or solo LTRs. For example, the
most arid sites were found to have both the highest copy
number of full length elements and the lowest number of
solo LTRs. Such results suggest that under stressful
conditions imposed by aridity, BARE-1 activity may be
triggered (possibly via epigenetic modifications) while
recombination may be reduced. It may be particularly rele-
vant to note that abscissic acid (ABA) response elements,
which are typical of water-stress induced genes, are present
J. Greilhuber and I.J. Leitch
within the BARE-1 promoter (Suoniemi et al. 1996b)
providing further support for the suggestion that water stress
may, in part, be responsible for the observed BARE-1 prolif-
eration in the most water-stressed sites on the SFS and that
BARE-1 may have a function for stress resistance and be
under selection.
Nevertheless, it is noted that despite differences in copy
number of BARE-1 between the study sites, there was no
evidence of significant intraspecific differences in genome
size between the six sites, although pooling the data for NFS
and SFS showed that the mean genome sizes between these
two slopes were significantly different, with the larger genomes
found in the SFS sample. It seems that at this microscale, other
repetitive elements within the genome may also be fluctuating
in numbers to compensate for the differences in copy number
of BARE-1 (Kalendar et al. 2000).
20.5.1.2 Ceratonia siliqua, Lotus peregrinus,
Cyclamen persicum
To investigate to what extent the results of H. spontaneum
represent a more general response to changes in microcli-
mate at Evolution Canyon I, several additional studies have
been conducted on plants with contrasting morphologies and
growth characteristics.
The first of these was by Bureš et al. (2004) who
investigated the carob tree Ceratonia siliqua (Fabaceae).
Like H. spontaneum, C. siliqua is also common in Evolution
Canyon I but exhibits a very different life strategy. In con-
trast to H. spontaneum, which is a herbaceous annual grass,
C. siliqua is a long lived perennial tree. Using flow
cytometry Bureš et al. estimated genome sizes in 79 adult
trees and 44 seedlings from below the same trees. Overall,
genome sizes ranged 1.04-fold, while between the NFS and
SFS the difference was considerably less, although still
significant, with genome sizes differing just 0.9% (d.f. ¼
77, p < 0.001). As observed for H. spontaneum, the south-
facing (“African”) slope exhibited the larger genomes.
Indeed, using multiple regression analysis, the most impor-
tant factor for genome size variation was shown to be slope
orientation (north- or south-facing) (p ¼ 0.0007), followed
by tree trunk circumference and leaf length (p ¼ 0.06).
Interestingly, seedlings did not show significant differences
in C-values between slopes, suggesting the possibility of
microclimatically-mediated selection, with the more stress-
ful arid sites on the SFS favoring, directly or indirectly,
larger genomes.
A second study was conducted by Gasmanová et al.
(2007), this time on the annual, mostly inbreeding bird’s
foot trefoil Lotus peregrinus (Fabaceae). Genome sizes
were estimated in 498 individuals (representing 10
genotypes) collected from six sites along the 300 m transect
of Evolution Canyon I. Like previous studies, intraspecific
variation was detected with C-values ranging 1.13-fold, and
20 Genome Size and the Phenotype
the mean C-value for the arid-stressed SFS (2.549 pg/2C)
was shown to be larger than the mean value for the NFS
(2.544 pg/2C). Nevertheless, Gasmanová et al. (2007) did
not find significant interslope differences in genome size,
based on the number of individuals analysed.
Most recently, Pavliček et al. (2008), examined yet
another contrasting growth form, a geophyte called the Per-
sian violet (Cyclamen persicum, Primulaceae), and once
again detected intraspecific variation in genome size,
although values were less variable, ranging only 1.06-fold
in the 75 samples analysed. They were unable to detect
significant differences in genome size between the two
slopes. Nevertheless, when the data were combined with
previous studies of C. siliqua, L. peregrinus and a beetle
(Oryzaephilus surinamensis, Sharaf et al. 2008), in a meta-
analysis a positive relationship between drought and genome
size was shown to be significant at Evolution Canyon I
(Pavliček et al. 2008).
Given the lack of extensive differences in genome size
of the plant species studied between the two contrasting
microsites of Evolution Canyon I, it remains to be deter-
mined to what extent genome size variation may be
adaptively significant or whether the subtle, and often non-
significant differences between the drier and more stressed
SFS and the wetter NFS simply represent correlations rather
than causations. To date it is certainly unclear how such
small differences in genome size could be visible to selective
forces unless selection is operating on one or more aspects of
genome size that are not yet recognized as being adaptively
relevant (Wendel and Wessler 2000). Indeed, it is likely that
different species will be under different selection pressures.
In a study of seven leaf characters (e.g., leaf size, leaf
thickness) from three woody species in Evolution Canyon I
(Olea europaea, Ceratonia siliqua and Pistacia lentiscus),
Nevo (2012) reported that all displayed drought resistant
adaptations on the SFS compared with the NFS, although
the adaptations differed between species. Of most relevance
to genome size was the length of the palisade cells (which
contributes to leaf thickness, with the thicker leaves charac-
teristic of more drought-adapted plants). The palisade cells
were significantly longer in plants of C. siliqua from the
more arid SFS compared with the NFS. Given the link
between genome size and cell size, it is perhaps tempting
to speculate that this is one possible place where selection of
genome size may be linked to drought adaptation at least in
C. siliqua. In the other two species, either the positive
relationship was only weakly significant (Pistacia lentiscus)
or the relationship was reversed (i.e., the palisade cells were
shorter in the SFS as in Olea europaea) or non-significant.
Such studies only serve to highlight further the complexity
of the interactions and indicate that additional studies are
clearly needed.
339
20.5.2 Festuca pallens
So far, intraspecific variation has been clearly demonstrated
by double peaks (see above) in four species of the fine-
leaved fescues of Festuca, i.e., F. pallens, F. polesica,
F. rupicola and F. vaginata (Šmarda 2006) but the most
significant and extensive studies have been made in F.
pallens which exibits two ploidy levels, diploids with
2n ¼ 14 and tetraploids with 2n ¼ 28 (Šmarda and Bureš
2006; Šmarda et al. 2007, 2008, 2010).
The initial investigations of F. pallens were conducted at
various geographical scales, ranging from the intrapopula-
tion level to surveys across the entire distribution range for
the species (Šmarda and Bureš 2006). Such studies uncov-
ered intraspecific differences of nearly 1.2-fold in genome
sizes in both diploid and tetraploid cytotypes. At the geo-
graphical scale, the larger genomes were observed to pre-
dominate in the southeastern part of their range, in contrast
to the northwestern populations which were generally
characterized by smaller genomes. Such patterns were
interpreted as reflecting the glacial history and post-glacial
migrations in the region rather than due to selection arising
from any environmental factors (Šmarda and Bureš 2006;
Šmarda et al. 2007).
Subsequent studies focused on 17 adult plants and 562
progeny and revealed that the range of genome sizes was
actually greater in the offspring produced in a single genera-
tion than in all the maternal plants studied (Šmarda et al.
2008). Even within a single panicle, the genome sizes of
their seedlings were shown to vary up to 1.117-fold. Clearly,
in F. pallens at least, intraspecific variation is dynamic. Not
only can it be generated very rapidly (i.e., in a single gener-
ation), but also it is apparent that continuous genome size
variation is being added to the population every generation.
The question as to whether such variation is under
stabilizing selection to minimize variation in established
adults was addressed by Šmarda et al. (2010). In a carefully
controlled experiment comparing the distribution of genome
sizes in a simulated competitive environment, Šmarda et al.
demonstrated that the genome size of F. pallens is
maintained by stabilizing selection for an ‘optimal’ genome
size, although it is weak given the persistent presence of
intraspecific variation in the adult plants. Indeed, further
studies are needed to determine whether a change in, for
example, environmental conditions, would result in a shift of
the ‘optimal’ genome size.
As to how such selection may be operating is currently
unclear. Indeed, the lack of observed correlations between
genome size and a range of phenotypic characters such as
seedling growth, measures of plant fitness, spatial location in
the test area, vegetation type or microclimatic conditions
(Šmarda et al. 2007, 2008) suggest that, at least in F. pallens,
340
selection on genome size is probably indirect and mediated
through some trait that interacts directly with the environ-
ment (Beaulieu 2010). It would appear therefore that at least
during the very initial stages of genome size diversification,
genome size may be non-adaptive (Gregory 2001;
Bennetzen et al. 2005).
20.6 Conclusions and Future Outlook
From the foregoing discussions, it is clear that there are an
impressive array of studies that have reported relationships
between genome size and phenotype at the nuclear, cellular
and whole plant levels and it is likely that more will be
uncovered in the future. Yet it must be borne in mind that
in nearly all cases, such relationships remain as correlations
with no clear understanding of the molecular and evolution-
ary forces underpinning the observed nucleotypic effects on
the phenotype. Indeed, as is clear from the studies of intra-
specific variation (Sect. 20.5), we are still grossly ignorant of
how changes in genome size are visible to selection and the
mechanisms operating which may lead to shifts in genome
size within and between species.
As we enter the next era of genomics, characterized by
the overwhelming amount of DNA sequence data being
generated by next generation and third generation sequenc-
ing technologies, it seems likely that there will be advances
in understanding genome size dynamics at the molecular
level and the evolutionary forces which may be responsible
for generating the diversity of genome sizes observed (e.g.,
Novák et al. 2010; Kelly and Leitch 2011; Macas et al. 2011;
Kelly et al. 2012). Linking this through to the whole plant
and phenotypic level will however, require additional under-
standing and insights from the fields of ecology and physiol-
ogy as noted by Cavalier-Smith ( 2005). In addition, the extent
to which phylogenetic history plays a role in determining how
shifts in genome size at the molecular level are translated into
phenotypic effects will be essential. Already there are hints
that such phylogenetic effects may be important players. For
example, comparisons of genome size diversity across angio-
sperm families show huge differences between them, with
some characterized by extensive variation (e.g., Orchidaceae,
Melanthiaceae) while others have much narrower ranges of
genome size despite good species representation (e.g.,
Brassicaceae, Lysák et al. 2009). In addition, the observation
that species with truly enormous genomes are phylogen-
etically clustered to just two orders of monocots (Liliales and
Asparagales) and one order of eudicots (Santalales) within the
angiosperms (Leitch et al. 2010; Leitch and Leitch 2013)
rather than being scattered across angiosperms suggests phy-
logenetic history may be an important component. The very
different genome size profiles observed between angiosperms
J. Greilhuber and I.J. Leitch
and gymnosperms (see Leitch and Leitch 2013, this volume)
may also, in part, arise from the fact that these two groups of
seed plants shared a last common ancestor c. 300 million years
ago and have since undergone very different evolutionary
trajectories. This is also reflected in their contrasting genome
dynamics (Leitch and Leitch 2012).
At the intraspecifc level, we are still grossly ignorant of
many aspects of this source of genome size variation such as
the factors which effect how often, how quickly and how much
intraspecific variation may be generated. In addition, the influ-
ence of selection forces, population size and age are also
poorly understood and need further investigation (Šmarda
and Bureš 2010). Given the improvements in flow cytometry
sensitivity, the tools are now at hand to enable at least some of
these unknowns to be addressed to give us a more comprehen-
sive view of the role intraspecific variation plays in generating
the diversity of genome sizes observed across plants.
Since ultimately the phenotype of a plant will determine
where, when and how a plant may grow, it is essential that
the role genome size plays in influencing the phenotype is
more fully appreciated and understood, especially if we are
to predict how plants will respond to the rapidly changing
world affected by climate change, increasing CO2 levels and
pollution. As discussed above, the limited studies that have
investigated this indicate that genome size may well be
important (e.g., see Sects. 20.4.5, 20.4.6). Nevertheless,
taken together, they also highlight the complexity of
interactions between genome size, phenotype, and a plant’s
response to environmental change. At the very least, such
studies emphasize the need to obtain and incorporate
genome size data into computer models and laboratory
experiments aiming to understand how plants will respond
to the many facets of environmental change induced through
human activity.
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 Index

A Avena (Oat), 14, 24, 25, 159, 325, 328

Aberrant mRNAs, 72 Azolla, 170, 312–313
Abies, 234, 237–239, 329
Abscisic acid (ABA), 106, 338
Acacia, 333 B
Adiantum capillus-veneris, 248 Bacteria, 113, 176, 326–327, 329
Aetanthus, chromosomes, 316 Bacterial artificial chromosomes (BACs), 2, 221–222
AFLPs. See Amplified fragment length polymorphisms (AFLPs) chromosome-specific, 14, 15, 176
Agmatoploidy, 195–199, 201, 203, 215 BARE-1, 19, 338
Algae, 4, 79, 82, 84, 189, 195, 256–258, 263, 328–330 Barley (Hordeum), 14, 19, 24
Allium, 14, 23, 100, 126, 152, 156, 212, 220, 264, 329, 332 Bayesian analysis, 2, 4
Allopolyploidy, 17, 19, 21–23, 25, 26, 213, 256–259, 267, 269, B chromosomes, 217, 235, 326, 327
270, 295, 298, 300. See also Polyploidy centromere, 150–152, 157–158, 160
Amniotes, 336 chromatin, 151–154, 157
Amphibians, 336 drive (mechanism), 155–158
Amplified fragment length polymorphisms (AFLPs) evolution, 149–160
analysis of allopolyploids, 22 genes, 149–152, 154–156, 158–160
analysis of B chromosomes, 155 influence on chromosome pairing, 217
analysis of ferns, 248 influence on diploidization, 154, 217
analysis of sex chromosomes, 177, 182 influence on cell cycle time, 326
Anaphase promoting complex (APC), 56, 78, 81, 83, 92, 104, 106, 132 occurrence, 217
Anamniotes, 336 origin, 150, 151, 158–160, 217
Aneuploidy, 86, 114, 129, 159, 191, 196–197, 203, 212–213, 297, 299 segregation, 150, 153, 155–158, 160
Angiosperms, 1–8, 100–101, 209–222, 255, 257–261, 269–270, transcription, 151, 153–155, 159
323–325, 333, 335–336, 340 Bigelowiella natans, 329
ancestral genome size, 8, 320 Bimodal karyotype, 197, 210, 216, 237
chromosome diversity, 209–222 Bivalent, 23, 126, 129, 131, 156, 192–194, 213–214, 235, 240, 250,
genome size diversity, 8, 315–317 267, 318
genome size evolution, 307–309, 314–319 Bixa orellana, 331
impact of polyploidy, 6–8, 213–214, 285–288 Botrychium, 308
mechanisms of genome size change, 317 Brachypodium distachyon, 20, 143, 210, 222
radiations, 6–7 Brassica, 14, 21, 71, 103, 108, 110, 112–113, 124, 126, 130, 131, 213,
sex determining systems, 171–179 278, 281–282
Antheridiogen, 169–170 Brassicaceae, 14, 21, 71, 103, 108–110, 112–113, 124, 126, 130, 131,
Apomixis, 267–269 141, 195, 211–214, 216, 221, 278, 281–282, 285, 319, 340
F1 hybrids, 23 Brassinosteroids, 106
polyploidy, 268–269 Breeding systems, 168–179, 204, 222, 246, 266, 269–270, 299,
Arachis hypogaea, 337 329–330
Arceuthobium, 316 Briza, 332
Artemisia, 332 Bromo-uridine, 68
Asparagales, 219–220, 317–318, 340 BrUTP, 69
Asteraceae, 7, 101, 150, 210–212, 217–220, 264, 285, 288, 336 Bryonia dioica, 178–179
Atkinsonia, 316 Bryophytes, 255, 257–258, 296
Aurora kinases, 87, 89–91 evolutionary relationships, 5, 307, 309
Autopolyploidy, 20, 21, 109, 213, 256, 258, 262, 267, genome size, 309–312, 319–320, 331
269–271, 295, 298 poikilohydry, 296, 301
Auxin, 85, 106, 107, 178, 287 polyploidy, 257–258, 296, 298–299, 319

I.J. Leitch et al. (eds.), Plant Genome Diversity Volume 2, 345

DOI 10.1007/978-3-7091-1160-4, # Springer-Verlag Wien 2013
346 Index

Bryophytes (cont.) chromatin remodelling, 84, 89, 129, 131, 151–154, 325
retrotransposons, 319 diminution, 201
sex chromosomes, 168–169 elimination, 24–26
vegetative reproduction, 299 epigenetics, 15, 16, 70, 89, 100, 102, 151–153, 194, 282
euchromatin, 16, 58, 70, 151, 153–154, 158–159, 194, 218, 325
heterochromatin, 16, 38, 40, 58, 66, 68, 70–71, 104, 130, 143, 151,
C 153–154, 156, 158, 176, 179, 189, 194,204, 214, 216, 218–219,
Cajal bodies, 66, 70, 73 236, 282, 310, 325–327
Cannabis sativa, 168, 177, 181, 221 loop, 36, 40, 59
Carica papaya, 168, 175, 177, 181, 221, 278, 280 organization, 15–16, 41, 46, 49, 58, 108, 115, 215
Carnivorous plants, 331 polymer melt, 39–41
Cations and chromosome condensation, 38–39 structure, 35–36, 38–41, 108, 127, 129–131, 187
C-bands, 189, 194, 214, 218, 234, 236 Chromocentres, 14, 15, 101, 104, 108
CDC25, 80, 82, 84 heterochromatic, 15, 108
CDKs (Cycline dependent kinases), 55, 77, 88, 90–92, 102–107, 125, Chromomycin A3 (CMA), 179, 236
128, 130–132 Chromosomes, 100–103, 106, 108, 114, 115, 245–248, 250, 251
Cell cycle, 14–15, 21, 55, 100–107, 110, 112, 324–327 alien segments, 14
CENH3 levels, 190 alien transcripts, 16
core cell cycle genes, 80–81, 83 ancestral base chromosome number, 7, 211, 213, 232, 299
cytokinins, 107 banding patterns, 25, 151–153, 189, 214, 217–219,
duration, 89, 325–328, 332 235–236, 239, 240
dynamics of the nuclear envelope, 55–57 condensation, 35–38, 41, 55, 90, 130, 131, 151, 153, 191–192, 235
LINC complexes, 47 deletions, 137–142, 144, 145, 196, 217
meiotic cell cycle, 128, 131–132 duplications, 137–141, 143–145
meiotic recombination, 132 elimination in F1 hybrids, 23–25
monocots versus eudicots, 326 evolution, 158–160, 195–202, 210–223, 231–241, 245–251,
nucleolus dynamics, 70–71 285–288, 298–300
phenology, 331–332 insertions, 137–140, 143–145
regulation, 77–92 inversions, 137–141, 143–144, 202, 212, 215, 222, 240
RNA processing, 132 landmarks, 214
transition to endocycles, 83–86, 102–104 monocentric versus holocentric, 187–192, 195, 202
Cell differentiation, 49, 79, 81, 83–86, 99–101, 107, 110, 113, 179 origin of eukaryotic chromosomes, 202
Cell size, 99, 112, 113, 259, 260, 270, 324–330 painting, 25, 26, 151, 160, 174, 217–218, 221
Centaurea, 264, 333 pairing, 23, 127–129, 130–131, 138, 140, 144, 154, 160, 196, 217,
Ceratonia siliqua, 338–339 222, 240, 246, 267, 318
Centric fusions/fissions, 7, 137, 141–144, 212, 215, 216, 240 pericentric inversions, 139–141, 143, 212, 215, 218
fusion, 141–142, 192, 195–199, 201–203 rate of chromosome change, 250
fission, 142–144, 192, 195–198, 200–202 reciprocal translocations, 137–141, 143–144, 157, 212, 216
holocentric chromosomes, 192, 195, 196, 198, 200–202 scaffold, 35–36, 38, 40
Rumex, 176 size, 23, 26, 140, 195, 198–199, 214, 311, 313
Centromeres, 14, 23–26, 38, 143–145, 175, 100, 212, 215, 222 structure, 35–36, 38–41
B chromosomes, 150–152, 157, 158, 160 territories (CTs), 14–15, 20, 23
CENH3, 24, 145, 151, 188, 190–191, 202–203 total chromosome length, 214
drive, 203–204 types, 187, 215
gymnosperms, 236, 237, 239 variation in number, 211
inactivation, 140, 143–145 Cohesins, 25, 78, 188, 191–192
neocentromeres, 144, 145, 188, 215 Collinearity analysis, 280
organization, 14, 26, 40 Collinsia verna, 337
position, 145, 215 Comparative mapping, 143, 150, 222, 240
recombination, 125 Concerted evolution, 22, 220, 301
repositioning (CR), 144, 145 Condensins, 36–37, 40, 78
sex chromosomes, 175–177 Copy number variation (CNV), 18
Centromeric repeats, 176, 215, 221 Cotton (Gossypium), 22–23, 130–131, 214, 220, 278, 281–282, 284
Ceratodon purpureus, 299, 302 Crossovers (COs), 121–122, 139–140
Ceratopteris richardii, 169–170, 247–250, 318 anti-crossover activity, 124
Chiasma, 23, 214 chromosome rearrangements, 139–141
B chromosomes, 155–156 correlation with synaptonemal complex, 126–127
in holocentric chromosomes, 192–194 factors affecting distribution, 125–127, 192
in polyploids, 129 formation of Class I COs, 123–124, 127–129
Chlorarachneans, 328–329 formation of Class II COs, 123, 127
Chlorophytes, 4 heterochiasmy, 126
Chromatin, 23, 33–35, 50, 55, 57, 59, 65, 78, 89, 151, 175, 187, holocentrics, 191–194, 203
189–191, 201, 214, 326 impact of GC content, 125
30-nm chromatin fibre, 34–36, 38–41 impact of polyploidy, 129–130
Index 347

interference, 125, 127 Cyclins (CYCs), 55, 78–89, 102–114, 132

number, 125, 127, 129–130, 130, 192–194, 203 Cytokinesis, 48, 54, 56, 77, 87, 91–92, 202
Ph1 locus, 130–132 Cytokinins, 103, 106–107
PrBn locus, 131
sex differences, 125–126
suppression between homoelogous chromosomes, 130–131 D
Cryptomonads, 328–329 DAPI. See 4’,6-Diamidino-2-phenylindole (DAPI)
Cucumis, 102, 110–111, 171–172, 222 Dasypyrum villosum, 337
C-values, 15, 19, 21, 23–24, 26, 108–109, 113, 115, 195, 198–200, 214, Dendrophthora, 316
232–323, 325–328, 331–333, 335–336, 338–340 Dense fibrillar component (DFC), 67–70
altitude, 333–334 dHJs. See Holliday junctions, double (dHJs)
angiosperms, 8, 307–309, 314–319 4’,6-Diamidino-2-phenylindole (DAPI), 103, 104, 108, 111, 236
aridity, 337–338 Dicentric, 139–143, 145, 151, 196, 215, 240
biomass, 329 Dioecious, 167–181
blood cells, 325, 329 Diploidisation, 18, 21, 25, 129, 144, 154, 213–214, 219, 250, 256, 259,
breeding systems, 329–330 296, 319, 325–326, 330
carbon dioxide (CO2), 334, 340 Diversification, 260–262
cell cycle duration, 325–328, 332 Diversity array technology (DArT), 26
cell size, 99, 112–113, 259–260, 270, 324–331, 325, 339 DNA damage checkpoint, 78
duration of meiosis, 326 DNA replication, 16, 40–41, 49, 58, 79, 87, 100, 102–107,
duration of mitosis, 326 130–132, 308
early diverging angiosperms, 316 DNA synthesis, 77–78, 83, 85, 101, 107, 138, 326
endopolyploidy, 99,101–104, 109–115 Double strand breaks (DSB), 121–125, 138–140, 143–144. See also
enigma, 324 Crossovers (COs); Holliday junctions, double (dHJs)
evolution, 307–309, 311, 314–316 chromatin structure in meiosis, 129
extinction risk, 336 formation during meiosis, 121, 123–125, 128–129
gymnosperms, 260, 314–315, 317, 320 genes involved during meiosis, 123–125
heavy-metal, 334 histone acetylation, 129
holoploid (C-value definition), 308 repair, 123–125, 138–140
hornworts, 308, 311 Drive
impact of polyploidy, 317, 327 B chromosomes, 155–158
invasive species, 332–333, 335 centromere, 203–204
life style, 327–329, 330, 332, 338 holocentric, 204
liverworts, 309–311 meiotic, 188, 201, 203
lycophytes, 308, 311–312 Drosera, 189, 195, 197–200, 331
mechanisms of genome size change, 317–318 Duplication, 137–141, 143–145
metabolic costs, 320 Dysploidy, 137, 141–144, 196, 212–213, 215–216, 262
minimum generation time, 328 ascending, 143–144, 212
monilophytes, 308, 312–314, 318–319 bidirectional, 144
monoploid (Cx-value definition), 307–308 descending, 141–143, 212
mosses, 308, 310–311, 319, 331 mixed dysploidy, 212
paradox, 324
phosphate, 330–331, 334
precipitation, 333–334 E
radiation sensitivity, 335 E2F binding transcription factor (E2F), 78, 81, 83–89, 92, 105–107
range across land plants, 308 Embryogenesis, 110, 160
recombination, 317–319, 330, 327, 338 Embryophytes, 4–5, 323, 307
reproduction mode, 329–330 Endocycle, 100–112, 114. See also Endopolyploidy
retrotransposons, 199, 204, 215, 220, 239, 251, 282, 317–320, DNA replication, 103–105
323, 335, 338 genes involved, 80–81, 84–86
seed plants, 308 regulation of endocycles, 83, 86, 102–103
selection pressures, 312, 317, 320 regulatory proteins, 105
speciation rates, 336 Endomitosis, 99–100, 114
terminology, 307–308 Endopolyploidy, 15, 99–115, 325, 329
UV, 334 epigenetic changes, 108
vascular plants, 308 factors regulating endopolyploidy, 106–108
weeds, 332, 335 mean C-level, 114
Cycads, 5, 167, 231, 331 occurrence of endopolyploidy, 101, 108–113
chromosome evolution, 232, 234–237, 239–240 patterns of endopolyploidy, 101–102, 109, 110
genome size, 314 structure of endopolyploid nucleus, 108
sex chromosomes, 170–171 types of endopolyploidy, 99–101
Cyclamen persicum, 338–339 Endoreduplication, 99–100. See also Endopolyploidy
Cycle value, 114–115 Endosymbiosis, 328
Cycline dependent kinases (CDK), 55, 77, 88, 90–92, 102–107, Environmental sex determination (ESD), 169–170
125, 128, 130–132 Ephedra, 231–232, 234–235, 257, 259–260, 263, 314–315, 317–318
348 Index

Ephemeral species, 325, 327–328, 335 Gymnosperm DNA C-values database, 232
Equisetum (horsetails), 308, 312–314 mutliflagellate sperm, 331
Evolution Canyon, 337–339 occurrence of paleopolyploidy, 318
Exon-junction complex (EJC), 72, 74 occurrence of polyploidy, 232, 259–260, 263, 268, 317
repetitive DNA, 237–239
retrotransposons, 239, 318
F ribosomal DNA, 237–238
Fabaceae, 109, 211–212, 216, 220, 264, 285, 288, 333, 338–339 sex chromosomes, 170–171
Ferns and allies. See Monilophytes Gynodioecy, 168, 176, 179
Festuca, 25–26, 323, 339
Festulolium, 25–26
F1 hybrids, 19, 21, 23–25, 236, 240 H
Fibrillar centres (FC), 66–67 Haploidization, 299
Fitness, 154, 179, 203, 216, 261, 263, 268, 270, 283, 330, 340 Helianthus (sunflower), 21, 103, 114, 220, 278, 337
Flow cytometry, 109, 111, 114, 115, 323, 332, 337, 338, 340 Helitrons, 18, 319
Fragaria, 168 Hermaphroditism, 167–169, 171–172, 176–178, 189, 203, 270, 299
Fritillaria, 317, 323, 331 Heterogametic, 125–126,168, 175, 177, 235
Funaria hygrometrica, 297, 299, 302 Heterosporous ferns, 170, 245–246, 259
Funariaceae, 296, 298–302 HiC procedure, 14–15
Histones, 15–16 33–35, 37–39, 84
Histone monoubiquitination 1 (HUB1), 88–89
G Histone phosphorylation, 37, 152, 190
Gaiadendron, 316 Histone modifications, 16, 23, 71, 78, 84, 152–154
gamma-tubulin (g-tubulin), 89, 92, 129, 301 B chromosomes, 153–154, 159
GC-content, 125, 327 CENH3, 24, 145, 151, 188, 190–191, 202–203
Gender dimorphism, 269–270 holocentric chromosomes, 190–191
Gene meiosis, 129
copy number variation (CNV), 18 nucleolar dominance, 71
conversion, 112, 125, 131, 141, 219, 280, 284–285, 300–301 polyploidy, 281
housekeeping, 17, 19 ribosomal DNA structure, 70
imprinted, 16–17, 71, 81 sex chromosomes, 175–179
nurseries, 18 Holliday junctions, double (dHJs), 123–125, 327
Presence or absence variation (PAV), 17–19 resolution in meiosis, 123
R-gene, 17, 18 Holocentric or holokinetic chromosomes, 107–202, 215
speciation genes, 17 evolution, 202–204
silencing, 16–17, 20, 22–24, 71, 73, 154, 177, 219, 247, 250–251, geographic distribution, 189
259, 282, 319, 326 meiosis, 191–194
Genetic linkage mapping, 248 recognition, 187–188
Genetic maps, 240 retrotransposons, 195–196, 199, 202, 204
Genlisea aurea, 137, 214–215, 309, 323–324, 331 verification, 187–192, 194, 196, 199
Genome Homogenization. See Gene conversion
diploidization, 18, 21, 25, 129, 144, 154, 213–214, 219, 250, 256, Homologous recombination (HR), 121, 125, 131, 138, 140,
259, 296, 319, 325–326, 330 260, 301, 319
drift, 25–26 Homoploid hybrids, 20, 211, 268, 271, 297–298
downsizing, 317–318, 327 Homosporous ferns, 169–170, 245–251, 259, 312, 318
elimination, 22–23, 35–36, 318–319, 326 Hordeum (barley), 14, 19, 24, 338
interaction, 22–23, 216, 282–284, 286 Hornworts, 5, 168, 309, 319
separation, 23–24 chromosome diversity, 308, 311
size (see C-value) genome size diversity, 308, 311
Gibberellic acid, 106–107 role of polyploidy, 257–258, 296, 311
Ginkgo biloba, 5, 167, 170, 231–233, 235–238, 257, 259, 314, 331 sex chromosomes, 168
Glaucophyta, 256–257, 272 Horsetails (Equisetum), 308, 312–314
Glycine, 278, 282, 333, 337 HR. See Homologous recombination (HR)
Gnetum (Gnetaceae), 231–232, 234–235, 259–260, 314–315, 318 Humulus lupulus, 168, 177, 181, 221
Gossypium (cotton), 22–23, 130–131, 214, 220, 278, 281–282, 284 Huperzia, 257, 259, 312
Granular component (GC), 66–67 Hybrid
Green algae, 4, 84, 189, 256–258, 263 necrosis, 17–19
Guard cell, 112, 325, 329 vigour, 19, 25–26
Guillardia theta, 329 Hybridization, 129, 159, 212–213, 217, 220, 240, 262–263, 266–269,
Gymnosperms, 5, 100, 114, 167, 170–171, 231, 255, 257, 259–260, 282–283, 297–300, 302
263, 268, 319–320, 329, 331–332
breeding systems, 170–171, 329
chromosome diversity, 231–240, 251 I
evolutionary relationships, 5, 307–308 Idiogram, 214
genetic map, 240 Imprinting, parental, 16
genome size diversity, 260, 314–315, 317, 320 Inbreeding, 168, 217, 203, 222, 246, 266, 269–270, 299, 329–330, 338
Index 349

Increased level of polyploidy1, 87 M

Integral membrane proteins, 46–47, 58–59 Maize (Zea mays), 13, 14, 16, 18, 19, 21, 24, 26, 38, 46, 48, 65, 79,
Intermediate filaments, 47, 49, 58 107–109, 112, 124–125, 127–129, 133, 144–145, 150–151, 154,
Introns, 4, 16, 72–73, 175 157–159, 171–173, 190, 217, 251, 277, 280, 317, 326, 328, 333
Isoetes, 5, 170, 311–312, 331 Marchantia polymorpha, 168–169, 302
Isozyme analysis, 246–248, 259, 267 Marsilea, 170, 313
Maximum likelihood analysis, 2–4, 8
Meiosis, 214. See also Crossovers (COs); Double strand breaks
J (DSB); Recombination
Jumping NOR, 145 B chromosomes, 155–156
bouquet, 128
chromatin structure, 129
K chromosome motility, 128–129
Karyoplasmic ratio, 324 comparison with mitosis, 122, 127, 131–132
Karyotype, 187, 189, 195–201, 204, 209–223 control of meiotic progression, 132
ancestral karyotype, 221 cyclin dependent kinases (CDKs), 125, 128, 130–132
asymmetric karyotypes, 216 cyclins, 128, 132
bimodal, 197, 210, 216, 237 diakinesis, 126, 128, 140
evolution in angiosperms, 141, 209–223 diplotene, 126, 128, 140
gymnosperms, 232, 235–237, 239–240 duration, 127, 326, 328, 335
holocentric, 195–199, 200–204 genes involved in meiosis progression, 124, 128, 130, 132
length, 214 histone modifications, 129
monilophytes, 245–247 holokinetic meiosis, 187, 192–194, 200
orthoselection, 197–198, 201, 239–240 leptotene, 126, 128
rate of chromosome change, 222, 250 metaphase I, 122, 128, 131–132
rearrangements, 137–145, 195–201, 211–212, 220 metaphase II, 122, 128, 131–132
role of retrotransposons, 199, 220, 222 pachytene, 126, 128–129
sex chromosomes, 175, 221, 235 Ph1 locus, 130–132, 154
structure, 212 polyploidy, 129–131
symmetric karyotypes, 214–215 PrBn locus, 131
unbalanced karyotypes, 212 prophase I, 122, 125, 128
Kinetochore, 24–25, 55, 78, 89–91, 151, 158, 187–188, 190–194, role of histone modifications, 129, 152
196, 202–204, 215 sister chromatid cohesion, 37, 126, 128, 152, 158, 192
KS distributions/plots, 248, 250, 278–281, 300–301 sister chromatid separation, 78, 121–122, 158
K-T extinction event, 7, 278, 288, 296 stages of meiosis, 122
synapsis, 124–129
synaptonemal complex (SC), 126–128, 130, 191, 193
L telekinetic meiosis, 187, 192–194, 202
Lamin-like proteins, 46, 58 wheat, 130–132
Lentibulariaceae, 210, 214–215, 323 zygotene, 126, 128–129
Library hypothesis, 221 Melanthiaceae, 189, 197, 200, 214–215, 217, 317, 323, 340
Liliales, 5, 150, 189, 217, 317–318, 326, 340 Mercurialis annua, 168, 178
Liverworts, 296 Microchromosomes, 217
biflagellate sperm, 331 Microseris douglasii, 336–337
chromosome diversity, 302 Microsatellite, 175–176, 201, 239
genome size diversity, 309–310 Miller spreads, 66, 69
role of polyploidy, 296, 313 Minimum interaction hypothesis, 216
sex determination, 169–170 Minority cytotype exclusion, 266, 269
Lolium, 23, 25–26, 154, 326, 329 Mitotic checkpoint, 78, 84, 92
Long interspersed nuclear elements (LINEs), 38, 177 Monilophytes
Long terminal repeat (LTR) retrotransposons, 19, 38, 152, 159, 175, breeding systems, 246
190, 199, 220, 239, 251, 318–319, 338 chromosomes, 246, 250–251, 318
Ty-copia, 19, 175, 199, 220, 239, 251, 319, 338 diversity, 307, 312–314
Ty-gypsy, 152, 159, 190, 220, 239, 318 evolutionary relationships, 5, 308, 318
Loranthaceae, 316 genome size, 308, 312–314, 318–319
Lotus, 49, 211, 280, 339 hypotheses concerning chromosome numbers, 246–247
Lycophytes, 5–6, 245–247, 251, 255, 257, 259 paleopolyploidy, 247–251, 318
chromosome diversity, 245–246, 311–312 retrotransposons, 251, 319
genome size diversity, 311–312, 320, 331 role of polyploidy, 247–251, 257–259, 319
Isoetes genome size evolution, 311–312 sex determination, 169–170
Selaginella genome evolution, 251, 311–312 Monoecious breeding system, 167–172, 177–179, 259, 299
sex determination, 170 Mosses, 5, 308–309
350 Index

Mosses (cont.) structure, 66–67

cell cycle regulation, 79, 87 Nucleomorphs, 328–329
chromosome diversity, 298–300, 302, 308, 311 Nucleoporins, 47, 48, 52–53, 55, 57
genome size diversity and evolution, 310–311, 319, 331 Nucleoskeleton, 45, 47, 57–59, 328, 329
retrotransposons, 319 Nucleosome, 33–35, 39–41
role of polyploidy, 257–258, 263, 270, 277, 289, 296–302, 311, 319 Nucleotype, 327–329
sex determination, 168 Nuytsia, 316
Mylia, 309–310

O
N Oat (Avena), 14, 24–25, 159, 325, 328
NAHR (Non-allelic homologous recombination), 138, 140–141 Olea europaea, 339
Nested chromosome fusion (NCF), 143 Ophioglossum, 137, 245, 257, 259, 308, 313
NHEJ (Non-homologous end joining), 138, 140 2’-O-ribose methylation, 69, 73
Nitrogen-fixing symbiosis, 113 Orchidaceae, 212, 264, 340
Non-allelic homologous recombination (NAHR), 138, 140–141 Oryza (rice), 13–15, 17–18, 26, 46, 48, 68, 79–82, 123–125, 127, 190,
Non-histone chromosomal proteins, 37 214, 251, 277–280, 284–285, 301, 317, 319
Non-histone proteins, 35–38 Oryzaephilus surinamensis, 339
Non-homologous end joining (NHEJ), 138, 140
Non-polysomatic plants. See Endopolyploidy
Non-reciprocal translocation, 140–141, 144 P
Nonsense-mediated mRNA decay (NMD), 72–73 p53, 74
NoRC (Nucleolar remodelling complex), 71 Paleopolyploidy (ancient WGDs), 231, 247–248, 250, 178–280, 297,
NOR (Nucleolar organizing region, NOR), 15, 24, 65–66, 68, 70, 108, 299, 302, 319
141, 143, 145, 216, 218–219, 239 Palisade cells, 339
Notothixos, 316 Paris japonica, 137, 214–215, 309, 317, 323, 326
Nuclear envelope, 15, 45–59, 66, 89, 100, 128 Paris quadrifolia, 326
cell cycle dynamics, 55–57 PAV (presence or absence variation), 17–19
comparison with other organisms, 50 Pearl millet, 24, 25
ion channels, 48–49 Pellia, 309–310
proteins, 46–50 Pericentric inversions, 139–141, 143, 212, 21
Nuclear membrane Perinucleolar compartment, 74
inner nuclear membrane, 46, 51, 59 Permanent translocation heterozygotes, 222
outer nuclear membrane, 45, 47–48, 52, 59 Ph1 locus, 130–132, 154
Nuclear organization, 13, 15, 17–20, 23 Phalaris arundinaceae, 332–333
Nuclear pore complex, 45–46, 50–57 Phoenix dactylifera, 179
comparison with other organisms, 57 Phoradendron, 316–317
nuclear pore assembly complex, 55–57 Phylloglossum, 312, 331
nuclear pore disassembly complex, 55–56 Physcomitrella patens, 296, 301
nucleocytoplasmic transport, 53–55 cell cycle, 79–81
nucleoporins, 47, 48, 52–53, 55, 57 chromosomes, 298–300
Nucleo-cytoplasmic ratio, 324–325, 328 classification, 300
Nucleolar organizing region (NOR), 15, 24, 65–66, 68, 70, 108, 141, genome size, 319
143, 145, 216, 218–219, 239 genome structure, 300–301, 319
pseudo-NORs, 68 helitrons, 319
Nucleolar remodelling complex (NoRC), 71 hybridization, 296–298
Nucleolar vacuole, 66 polyploidy, 277, 296–301, 319
Nucleolus, 14, 15 retrotransposons, 319
assembly and dynamics of nucleolus, 70 Picea, 152, 232, 234, 237–239, 241, 329
‘Christmas trees’, 66 Pilularia, 313
dynamic studies, 70 Pinus, 48, 114, 170, 239–240, 329, 332
functions, 74 chromosome diversity, 232–236, 240
non-conventional roles of nucleolus, 71–74 fossils, 240
nonsense-mediated mRNA decay (NMD), 72–73 genetic maps, 240
nucleolar dominance, 70–71, 159, 219 invasiveness, 332
organiser region(s) NORs, 15, 65, 108, 216 retrotransposons, 239
perinucleolar compartment, 74 ribosomal DNA, 237–238
pre-nucleolar bodies, 70 telomeres, 237
refractive index, 66 transcriptome, 239
residence time in the nucleolus, 70 Pistacia lentiscus, 339
ribosome production rate, 65 Pistia stratiotes, 332
RNA modifications, 69, 73 Plant DNA C-values database, 308, 315, 332
role in mRNA export, 72–73 Poa annua, 337
role in translation, 73 Polo-like kinase, 89
siRNA production, 73 Polymer melt, 39–41
Index 351

Polymorphisms, 13, 16, 17, 23 Pseudoalleles, 301

Polyploidy, 20, 22, 109–111, 113–114, 137, 195–196, 199–201, Pseudouridylation, 69, 73
212–213, 232, 247–248, 251, 295–297, 299–300, 302, Psilotum
307–308, 311, 313, 315–320, 323, 326, 328, 330–331, 333, chromosome evolution, 308
335–337 genome size, 312, 313
adaptive potential of novel polyploids, 270–271, 283–284 retrotransposons, 319
asexual reproduction, 268–269 Psittacanthus, 316, 317
autopolyploid genome evolution, 20–21, 109, 295
changes in expression pattern, 21–23, 282–283 R
chromosome changes, 23, 25–26, 137, 144, 212–214 Rabl organisation, 14–15, 40, 91
coexistence of cytotypes, 265–266 Ralstonia solanacearum, 176
comparison with animals, 261–262, 268 Ranunculaceae, 101, 109, 177, 211, 218, 264, 331
dating ancient polyploid events, 278, 280–281 Ranunculus ficaria, 331
detecting ancient polyploid events, 247, 248, 278–280 Ranunculus adoneus, 264
diploidisation, 18, 21, 25, 129, 144, 154, 213–214, 219, 250, 256, Reciprocal translocations, 137–141, 143–144, 157, 212, 216
259, 296, 319, 325–326, 330 Recombination, 23, 25–26, 121–127, 138, 167, 248, 310, 318, 327, 330,
dosage balance effects, 286 338. See also Crossovers (COs); Holliday junctions, double
epigenetic changes, 21–22, 281–284 (dHJs), and Double strand breaks (DSB)
‘Escape from adaptive conflict’ (EAC) model, 287 anti-crossover activity, 124
establishment, 255, 260, 267–270, 272 B chromosomes, 150, 217
evolutionary novelty, 286–287 Brassica, 124, 130–131
evolutionary potential, 287–288 centromeric recombination, 125
formation, 255–256, 260–263, 267–271 choice of template, 125, 132
gene conversion, 284 chromatin structure, 129
gene loss, 21–22, 285–286, 300 chromosome rearrangements, 138, 140–141, 145, 160
gene silencing, 247, 251 cotton, 131
genetic changes, 281 distribution, 125, 129, 131
genome divergence, 256, 261, 271, 284–285 gene conversion, 121, 131, 284
genome homogenization, 284 genetic control, 130–132
geographic distribution, 259, 263–265, 270–271 genome size, 317–319, 327, 338
holocentric chromosomes, 195–196, 199–201, 215 histone modifications, 129, 152
impact on degree of endopolyploidy, 109 holocentric chromosomes, 191, 203
Increased Level of Polyploidy1 (ILP1), 87 hybrids, 26
‘Innovation, amplification, divergence’ (IAD) model, 287 illegitimate recombination, 138, 250, 317
intraspecific variation in ploidy level, 212, 262–267 impact on phylogenetic studies, 4
KT extinction event, 7, 278, 288, 296 meiotic recombination, 121–127, 138, 141
life form, 267–268 non-allelic homologous recombination (NAHR), 138, 140–141
mating system, 178, 269–270 non-crossovers (NCOs), 123, 131
meiosis, 129–131 Ph1, 130
mode of origin, 213–214, 267 polyploidy, 20, 129–130, 284
occurrence in angiosperms, 213, 260 PrBn, 131, 132
occurrence in bryophytes, 258, 296, 298 recombination nodules, 127
occurrence in ferns, 245–251, 258–259 sex chromosomes, 169, 177, 180–181, 221
occurrence in glaucophytes, 256–257 wheat, 130–132
occurrence in green algae, 257–258 Red algae (Rhodophyta), 4, 256–257, 262, 328
occurrence in gymnosperms, 232–235, 259–260 Regnellidium, 313
occurrence in lycophytes, 257, 259 Regulatory spandrel, 286–289
preadaptation, 260, 269 Renner complexes, 222
range size, 270–271 Replication origins, 23, 77, 102
recombination, 20, 129–130, 284 Resistence genes (R-genes), 17
regulatory spandrels, 286–289 Retinoblastoma related protein (RBR), 78–79, 80–81, 83–84, 86–87,
retrotransposons, 21, 220, 282–284, 319 90, 103, 107
speciation, 7, 213, 260–261, 285, 295 Retrotransposons (retroelements), 19, 21, 24, 38, 150–151, 154, 159,
stability of polyploids, 260 175, 177, 190, 195–196, 199, 202, 204, 215, 220, 221, 222, 239,
subfunctionalization, 261, 285–286, 300 251, 282, 317–320, 323, 335, 338. See also Long terminal repeat
taxonomic variation, 256–261 (LTR) retrotransposons
transposable elements, 21, 220, 282–284, 319 Rhodophyta (Red algae), 4, 256–257
vegetative reproduction, 269 Ribosomal DNA (rDNA)/RNA (rRNA), 237. See also Nucleolus;
Polysomatic plants. See Endopolyploidy Nucleolar organizing regions (NORs)
Polytene chromosome, 100 B chromosomes, 151, 153–155, 159
Pre-nucleolar bodies, 70 evolution, 219
Presence or absence variation (PAV), 17–19 genes in gymnosperms, 237–238
Primary chromosome rearrangement, 138–141, 144 genes in monilophytes, 247
352 Index

Ribosomal DNA (rDNA)/RNA (rRNA) (cont.) SMC (Structural maintenance of chromosome) protein, 36–37, 106
holocentric chromosomes, 190 Sorghum, 14, 24, 48, 277–278, 280, 284–285
karyotype analysis, 219 Sperm
Miller spreads, 66, 69 biflagellate sperm, 312
mobility, 145, 219, 237 multiflagellate sperm, 312
number of copies 68, 145 relationship between C-value and flagella number, 331
organization of rDNA, 67–68, 151, 219, 237 Spinacia oleracea (spinach), 110, 177–178
rDNA transcription, 68–70, 151, 155, 159 Spindle, 48, 50, 55–56, 59, 78, 87, 89–92, 128, 156, 187–188, 191, 194,
ribosome biogenesis, 68–69 202–204, 209, 214–215
RNA mediated silencing of rRNA, 71, 73 B chromosomes, 156
Robertsonian translocations, 141, 143 chromosome elimination, 24
telomere synthesis, 143 holocentric chromosomes, 187–188. 191, 194, 202–204
transposable elements, 145, 239 meiosis, 128, 132, 203
upstream binding factor, 68, 70 nuclear envelope, 50
Ribosome production, 65 polyploidy, 260
Ribosome biogenesis, 68–69 spindle assembly, 56
Rice (Oryza), 13–15, 17–18, 26, 46, 48, 68, 79–82, 123–125, 127, 190, g-tubulin, 89–90
214, 251, 277–280, 284–285, 301, 317, 319 SSRs (Simple sequence repeats), 239
Ring chromosome, 139–140 Streptophytes, 4
RNA interference (RNAi), 155, 190 Structural maintenance of chromosome (SMC) protein, 36–37, 106
Robertsonian (Rb) translocation, 141–143, 203, 212 Struthanthus, 316–317
R1R2R3-type MYB transcription factors, 82, 87–89 SUMO (Small ubiquitin-like modifier), 105–106
R-strategists, 332 Sunflower (Helianthus), 21, 103, 114, 220, 278, 337
Rudbeckia laciniata, 332 Supermatrix, 5, 7
Rumex, 168, 176–177, 179, 181 Supernumerary segment, 211, 216–217
R. acetosa, 174–177, 182, 217, 221 Supertree, 5, 7
R. hastatulus, 176 Symploidy, 195–197, 199, 215
Synaptonemal complex (SC), 126–128, 130
structure, 126–127
S influence of crossovers, 126–127
Salvinia, 313 in holocentric chromosomes, 191–193
Santalales Synteny, 23, 240, 248, 284
chromosome evolution, 316–317
genome size diversity, 317, 340
phylogenetic relationships, 6, 316 T
Satellite DNA. See Tandem repeat DNA; Ribosomal DNA Tandem repeat DNA (= satellite DNA), 16, 38, 141, 215–218,
Secale, 150–151, 154, 147, 159, 218, 326 220–222, 235, 319. See also Ribosomal DNA
Secondary chromosome rearrangement, 141 B chromosomes, 150–152, 154–155, 158–159
Secondary constrictions, 65, 68, 70, 209–211, 214, 216, 219, 235 centromeric repeats, 176, 215, 221
Selaginella, 170, 246, 251 gymnosperm-specific satellites, 239
cell cycle genes, 79–81 holocentric chromosomes, 190, 201–204
chromosome evolution, 246, 251, 311 role in sister chromatid separation, 158
genome size, 311–312, 331 sex chromosomes, 176, 221
sex determination, 170 telomeric repeats, 143, 152–153, 219–220, 235, 237, 239
Senecio, 22, 210, 263–264, 282 Tasselseed1/Tasselseed2, 171–173
Sequoia, 231, 232–233,257, 259, 315 TE (transposable elements), 16, 19, 21, 23, 145, 214, 218, 251, 327,
Sex chromosomes, 168–182, 221, 235 330, 338
Sex determination, 168–173, 176–181 Telomerase, 73–74, 142–143, 219
Sexual dimorphism, 177–181 Telomeric repeats, 143, 152–153, 219–220, 235, 237, 239
Short interspersed nuclear elements (SINEs), 38 Telomeres, 14, 40, 18, 138, 142–143, 145, 190, 201, 219, 236
Signal recognition particle (SRP), 73 Thalictrum dioicum, 177
SILAC, 71 Tmesipteris, 308, 313
Silene, 168, 176, 221 Topoisomerase II, 35–37, 59
S. colpophylla, 174, 176 Topoisomerase VI, 103–106
S. dioica, 172, 174 Topoisomerase 3a, 124
S. latifolia, 172–175, 177, 179, 181–182 Transcription factors, 41, 57, 286
S. otites, 176 E2F, 78, 81, 83–89, 92, 103, 105–107
Silver-staining, 68, 216 Increased level of polyploidy1, 87
Simple sequence repeats (SSRs), 239 MADS-box, 287–299
Small interfering RNA (siRNA), 16–17, 19, 22, 71, 73–74, 319 p53, 74
Small nucleolar RNA (snoRNA), 69, 73–74 R1R2R3-MYB, 81, 87–89
Box CD snoRNAs, 69 RBR, 81
Box H/ACA snoRNAs, 69 TCP, 89
Small ubiquitin-like modifier (SUMO), 105–106 Transgressive and non-transgressive expression patterns, 284
Index 353

Transposable elements (TEs), 16, 19, 21, 23, 145, 214, 218, Viridiplantae (Green plants), 3, 4, 256, 257
251, 327, 330, 338 Viscum, 316, 323
Trillium, 127, 317, 323, 325–326
Triticum, 14–15, 21–22, 24–26, 40, 48, 68, 70, 102, 108, 127,
130–132, 151–152, 154, 157, 251, 277, 280–282, 326, 328 W
Ty-copia, 19, 175, 199, 220, 239, 251, 319, 338 WEE1 kinase, 80, 82–83, 92, 105
Ty-gypsy, 152, 159, 190, 220, 239, 318 Welwitschia, 231–232, 234–235, 259–260, 315
Whisk ferns, 258–259, 308, 312–313, 319
Whole genome duplications (WGDs). see Polyploidy
U
Upstream binding factor (UBF), 68, 70
X
X to autosome dosage system, 176, 221, 262
V
Vacuoles, 325, 328–329
Vegetative reproduction, 111, 156, 169, 267–269, 299 Z
Veronica, 330, 334 Zea mays. See Maize
Vicia, 14, 91, 141, 144, 189, 220, 263–264, 324–325

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